U.S. patent application number 14/772140 was filed with the patent office on 2016-03-10 for fungal resistant plants expressing mybtf.
This patent application is currently assigned to BASF PLANT SCIENCE COMPANY GMBH. The applicant listed for this patent is BASF PLANT SCIENCE COMPANY GMBH. Invention is credited to Ralf Flachmann, Tobias Mentzel, Holger Schultheiss.
Application Number | 20160068856 14/772140 |
Document ID | / |
Family ID | 47827076 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160068856 |
Kind Code |
A1 |
Schultheiss; Holger ; et
al. |
March 10, 2016 |
Fungal Resistant Plants Expressing MYBTF
Abstract
The present invention relates to a method of increasing
resistance against fungal pathogens of the order Pucciniales in
plants and/or plant cells. This is achieved by increasing the
expression of a MybTF protein or fragment thereof in a plant, plant
part and/or plant cell in comparison to wild type plants, wild type
plant parts and/or wild type plant cells. Furthermore, the
invention relates to transgenic plants, plant parts, and/or plant
cells having an increased resistance against fungal pathogens, in
particular, pathogens of the order Pucciniales, and to recombinant
expression vectors comprising a sequence that is identical or
homologous to a sequence encoding a MybTF protein.
Inventors: |
Schultheiss; Holger;
(Boehl-lggelheim, DE) ; Flachmann; Ralf;
(Limburgerhof, DE) ; Mentzel; Tobias; (Roemerberg,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF PLANT SCIENCE COMPANY GMBH |
Ludwigshafen |
|
DE |
|
|
Assignee: |
BASF PLANT SCIENCE COMPANY
GMBH
Ludwigshafen
DE
|
Family ID: |
47827076 |
Appl. No.: |
14/772140 |
Filed: |
March 7, 2014 |
PCT Filed: |
March 7, 2014 |
PCT NO: |
PCT/EP2014/054461 |
371 Date: |
September 2, 2015 |
Current U.S.
Class: |
800/265 ;
426/615; 435/320.1; 435/418; 435/468; 554/8; 800/279; 800/301 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8282 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2013 |
EP |
13158321.3 |
Claims
1. A method for increasing fungal resistance in a plant, a plant
part, or a plant cell, wherein the method comprises the step of
increasing the expression and/or activity of a MybTF protein in the
plant, plant part, or plant cell in comparison to a wild type
plant, wild type plant part or wild type plant cell and wherein the
MybTF protein comprises an amino acid sequence encoded by (i) a
nucleic acid having a nucleic acid sequence with at least 70%
identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, or a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; (ii) a nucleic acid encoding a protein having an
amino acid sequence with at least 70% identity with SEQ ID NO: 7,
5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or
56, or a functional fragment thereof, an orthologue or a paralogue
thereof; (iii) a nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); and/or by (iv) a nucleic
acid encoding the same MybTF protein as any of the nucleic acids of
(i) to (iii) above, but differing from the nucleic acids of (i) to
(iii) above due to the degeneracy of the genetic code.
2. The method of claim 1, comprising (a) stably transforming a
plant cell with an expression cassette comprising (i) an exogenous
nucleic acid having a nucleic acid sequence with at least 70%
identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, or a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; (ii) an exogenous nucleic acid encoding a protein
having an amino acid sequence with at least 70% identity with SEQ
ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, or 56, or a functional fragment thereof, an orthologue or a
paralogue thereof; (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii),
and/or (iv) an exogenous nucleic acid encoding the same MybTF
protein as any of the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code, in functional linkage with a
promoter; (b) regenerating the plant from the plant cell; and (c)
expressing said exogenous nucleic acid.
3. A recombinant vector construct comprising: (a) (i) a nucleic
acid having a nucleic acid sequence with at least 70% identity with
SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, or 55, or a functional fragment
thereof, an orthologue or a paralogue thereof, or a splice variant
thereof; (ii) a nucleic acid encoding a protein having an amino
acid sequence with at least 70% identity with SEQ ID NO: 7, 5, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, or a
functional fragment thereof, an orthologue or a paralogue thereof;
(iii) a nucleic acid capable of hybridizing under stringent
conditions with a complementary sequence of any of the nucleic
acids according to (i) or (ii), and/or (iv) a nucleic acid encoding
the same MybTF protein as any of the nucleic acids of (i) to (iii)
above, but differing from the nucleic acids of (i) to (iii) above
due to the degeneracy of the genetic code, operably linked with (b)
a promoter and (c) a transcription termination sequence.
4. The method of claim 2, wherein the promoter is a constitutive
promoter, pathogen-inducible promoter, a mesophyll-specific
promoter or an epidermis specific-promoter.
5. A transgenic plant, transgenic plant part, or transgenic plant
cell transformed with the recombinant vector construct of claim
3.
6. A method for the production of a transgenic plant, transgenic
plant part, or transgenic plant cell having increased fungal
resistance, comprising (a) introducing the recombinant vector
construct of claim 3 into a plant, a plant part, or a plant cell;
(b) generating a transgenic plant, transgenic plant part, or
transgenic plant cell from the plant, plant part or plant cell; and
(c) expressing the MybTF protein encoded by (i) the exogenous
nucleic acid having a nucleic acid sequence with at least 70%
identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; (ii) the exogenous nucleic acid encoding a protein
having an amino acid sequence with at least 70% identity with SEQ
ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, or 56, or a functional fragment thereof, an orthologue or a
paralogue thereof; (iii) the exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
and/or by (iv) the exogenous nucleic acid encoding the same MybTF
protein as any of the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code.
7. The method of claim 6, further comprising the step of harvesting
the seeds of the transgenic plant and planting the seeds and
growing the seeds to plants, wherein the grown plants comprise (i)
the exogenous nucleic acid having a nucleic acid sequence with at
least 70% identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27,
29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, a
functional fragment thereof, an orthologue or a paralogue thereof,
or a splice variant thereof; (ii) the exogenous nucleic acid
encoding a protein having an amino acid sequence with at least 70%
identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, or 56, or a functional fragment thereof, an
orthologue or a paralogue thereof; (iii) the exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); and/or (iv) the exogenous nucleic acid encoding the same
MybTF protein as any of the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
8. (canceled)
9. A harvestable part of the transgenic plant of claim 5.
10. A product derived from the plant of claim 5.
11. A method for the production of a product comprising a) growing
the plant of claim 5; and b) producing said product from or by the
plant and/or part of the plant.
12. The method of claim 11 comprising a) growing the plant and
removing the harvestable parts from the plant; and b) producing
said product from or by the harvestable parts of the plant.
13. The method of claim 11, wherein the product is meal or oil.
14. The method of claim 1, wherein the fungal resistance is
resistance against rust fungus, downy mildew, powdery mildew, leaf
spot, late blight and/or septoria.
15. The method of claim 14, wherein the fungal resistance is a
resistance against soybean rust.
16. The method of claim 15, wherein the resistance against soybean
rust is resistance against Phakopsora meibomiae and/or Phakopsora
pachyrhizi.
17. The method of claim 1, wherein the plant is selected from the
group consisting of beans, soya, pea, clover, kudzu, lucerne,
lentils, lupins, vetches, groundnut, rice, wheat, barley,
arabidopsis, lentil, banana, canola, cotton, potato, corn, sugar
cane, alfalfa, and sugar beet.
18. A method for breeding a fungal resistant plant comprising (a)
crossing the plant of claim 5 with a second plant; (b) obtaining
seed from the cross of step (a); (c) planting said seeds and
growing the seeds to plants; and (d) selecting from said plants
plants expressing a MybTF protein encoded by (i) the exogenous
nucleic acid having a nucleic acid sequence with at least 70%
identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; (ii) the exogenous nucleic acid encoding a protein
having an amino acid sequence with at least 70% identity with SEQ
ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, or 56, or a functional fragment thereof, an orthologue or a
paralogue thereof; (iii) the exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
and/or by (iv) the exogenous nucleic acid encoding the same MybTF
protein as any of the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code.
Description
[0001] This application claims priority to EP 13158321.3
application number filed Mar. 8, 2013 which is incorporated herein
by reference in their entirety.
SUMMARY OF THE INVENTION
[0002] The present invention relates to a method of increasing
resistance against fungal pathogens, in particular, pathogens of
the order Pucciniales, for example soybean rust, in plants, plant
parts, and/or plant cells. This is achieved by increasing the
expression and/or activity of a Myb-like transcription factor
(MybTF) protein in a plant, plant part and/or plant cell in
comparison to wild type plants, wild type plant parts and/or wild
type plant cells.
[0003] Furthermore, the invention relates to transgenic plants,
plant parts, and/or plant cells having an increased resistance
against fungal pathogens, in particular, pathogens of the order
Pucciniales, for example soybean rust, and to recombinant
expression vectors comprising a sequence that is identical or
homologous to a sequence encoding a MybTF protein.
BACKGROUND OF THE INVENTION
[0004] The cultivation of agricultural crop plants serves mainly
for the production of foodstuffs for humans and animals.
Monocultures in particular, which are the rule nowadays, are highly
susceptible to an epidemic-like spreading of diseases. The result
is markedly reduced yields. To date, the pathogenic organisms have
been controlled mainly by using pesticides. Nowadays, the
possibility of directly modifying the genetic disposition of a
plant or pathogen is also open to man.
[0005] Resistance generally describes the ability of a plant to
prevent, or at least curtail the infestation and colonization by a
harmful pathogen. Different mechanisms can be discerned in the
naturally occurring resistance, with which the plants fend off
colonization by phytopathogenic organisms. These specific
interactions between the pathogen and the host determine the course
of infection (Schopfer and Brennicke (1999) Pflanzenphysiologie,
Springer Verlag, Berlin-Heidelberg, Germany).
[0006] With regard to the race specific resistance, also called
host resistance, a differentiation is made between compatible and
incompatible interactions. In the compatible interaction, an
interaction occurs between a virulent pathogen and a susceptible
plant. The pathogen survives, and may build up reproduction
structures, while the host mostly dies off. An incompatible
interaction occurs on the other hand when the pathogen infects the
plant but is inhibited in its growth before or after weak
development of symptoms (mostly by the presence of R genes of the
NBS-LRR family, see below). In the latter case, the plant is
resistant to the respective pathogen (Schopfer and Brennicke, vide
supra). However, this type of resistance is specific for a certain
strain or pathogen.
[0007] In both compatible and incompatible interactions a defensive
and specific reaction of the host to the pathogen occurs. In
nature, however, this resistance is often overcome because of the
rapid evolutionary development of new virulent races of the
pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16
No. 7: 626-633).
[0008] Most pathogens are plant-species specific. This means that a
pathogen can induce a disease in a certain plant species, but not
in other plant species (Heath (2002) Can. J. Plant Pathol. 24:
259-264). The resistance against a pathogen in certain plant
species is called non-host resistance. The non-host resistance
offers strong, broad, and permanent protection from phytopathogens.
Genes providing non-host resistance provide the opportunity of a
strong, broad and permanent protection against certain diseases in
non-host plants. In particular, such a resistance works for
different strains of the pathogen.
[0009] Fungi are distributed worldwide. Approximately 100 000
different fungal species are known to date. Thereof rusts are of
great importance. They can have a complicated development cycle
with up to five different spore stages (spermatium, aecidiospore,
uredospore, teleutospore and basidiospore).
[0010] During the infection of plants by pathogenic fungi,
different phases are usually observed. The first phases of the
interaction between phytopathogenic fungi and their potential host
plants are decisive for the colonization of the plant by the
fungus. During the first stage of the infection, the spores become
attached to the surface of the plants, germinate, and the fungus
penetrates the plant. Fungi may penetrate the plant via existing
ports such as stomata, lenticels, hydatodes and wounds, or else
they penetrate the plant epidermis directly as the result of the
mechanical force and with the aid of cell-wall-digesting enzymes.
Specific infection structures are developed for penetration of the
plant.
[0011] Immediately after recognition of a potential pathogen the
plant starts to elicit defense reactions. Mostly the presence of
the pathogen is sensed via so called PAMP receptors, a class of
trans-membrane receptor like kinases recognizing conserved pathogen
associated molecules (e.g. flagellin or chitin). Downstream of the
PAMP receptors, the phytohormones salicylic acid (SA), jasmonate
(JA) and ethylene (ET) play a critical role in the regulation of
the different defense reactions. Depending on the ratio of the
different phytohormones, different defense reactions are elicited
by the host cell. Generally SA dependent defense is linked with
resistance against biotrophic pathogens, whereas JA/ET dependent
defense reactions are in most cases directed against necrotrophic
pathogens (and insects).
[0012] Another more specific resistance mechanism is based on the
presence of so called resistance genes (R-genes). Most R genes
belong to the nucleotide-binding site--leucine-rich repeat
(NBS-LRR) gene family and function in monitoring the presence of
pathogen effector proteins (virulence factors; avirulence factors).
After recognizing the pathogen derived proteins a strong defense
reaction (mostly accompanied by a programmed cell death) is
elicited.
[0013] The soybean rust Phakopsora pachyrhizi directly penetrates
the plant epidermis. After crossing the epidermal cell, the fungus
reaches the intercellular space of the mesophyll, where the fungus
starts to spread through the leaves. To acquire nutrients the
fungus penetrates mesophyll cells and develops haustoria inside the
mesophyl cell. During the penetration process the plasmamembrane of
the penetrated mesophyll cell stays intact. Therefore the soybean
rust fungus establishes a biotrophic interaction with soybean.
[0014] The biotrophic phytopathogenic fungi, such as soybean rust
and all other rust fungi, depend for their nutrition on the
metabolism of living cells of the plants. This type of fungi belong
to the group of biotrophic fungi, like other rust fungi, powdery
mildew fungi or oomycete pathogens like the genus Phytophthora or
Peronospora. The necrotrophic phytopathogenic fungi depend for
their nutrition on dead cells of the plants, e.g. species from the
genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has
occupied an intermediate position, since it penetrates the
epidermis directly, whereupon the penetrated cell becomes necrotic.
After the penetration, the fungus changes over to an
obligatory-biotrophic lifestyle. The subgroup of the biotrophic
fungal pathogens which follows essentially such an infection
strategy is heminecrotrohic. In contrast to a heminecrotrophic
pathogen, a hemibiotrophic pathogen lives for a short period of
time in a biotrophic manner and subsequently starts killing the
host cell and/or host organism, i.e., changes for the rest of its
life-cycle to a necrotrophic life-style.
[0015] Soybean rust has become increasingly important in recent
times. The disease may be caused by the biotrophic rusts Phakopsora
pachyrhizi and Phakopsora meibomiae. They belong to the class
Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts
infect a wide spectrum of leguminosic host plants. P. pachyrhizi,
also referred to as Asian rust, is the more aggressive pathogen on
soy (Glycine max), and is therefore, at least currently, of great
importance for agriculture. P. pachyrhizi can be found in nearly
all tropical and subtropical soy growing regions of the world. P.
pachyrhizi is capable of infecting 31 species from 17 families of
the Leguminosae under natural conditions and is capable of growing
on further 60 species under controlled conditions (Sinclair et al.
(eds.), Proceedings of the rust workshop (1995), National
SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J. L. et
al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the
Caribbean Basin and in Puerto Rico, and has not caused substantial
damage as yet.
[0016] P. pachyrhizi can currently be controlled in the field only
by means of fungicides. Soy plants with resistance to the entire
spectrum of the isolates are not available. When searching for
resistant soybean accessions, six dominant R-genes of the NBS-LRR
family, named Rpp1-5 and Rpp? (Hyuuga), which mediate resistance of
soy to P. pachyrhizi, were discovered by screening thousands of
soybean varieties. As the R-genes are derived from a host
(soybean), the resistance was lost rapidly, as P. pachyrhizi
develops new virulent races. Therefore there is a strong need to
discover R-genes that are derived from non-hosts plants (e.g.
Arabidopsis) as they are thought to be more durable.
[0017] In recent years, fungal diseases, e.g. soybean rust, has
gained in importance as pest in agricultural production. There was
therefore a demand in the prior art for developing methods to
control fungi and to provide fungal resistant plants.
[0018] Much research has been performed on the field of powdery and
downy mildew infecting the epidermal layer of plants. However, the
problem to cope with soybean rust which infects the mesophyll
remains unsolved.
[0019] The object of the present invention is inter alia to provide
a method of increasing resistance against fungal pathogens,
preferably rust pathogens (i.e., fungal pathogens of the order
Pucciniales), preferably against fungal pathogens of the family
Phacopsoraceae, more preferably against fungal pathogens of the
genus Phacopsora, most preferably against Phakopsora pachyrhizi and
Phakopsora meibomiae, also known as soybean rust.
[0020] Surprisingly, we found that fungal pathogens, in particular
of the order Pucciniales, for example soybean rust, can be
controlled by increasing the expression of a MybTF protein. The
MybTF described in this invention belongs to the R2R3-MYB
family.
[0021] The present invention therefore provides a method of
increasing resistance against fungal pathogens, preferably against
rust pathogens (i.e., fungal pathogens of the order Pucciniales),
preferably against fungal pathogens of the family Phacopsoraceae,
more preferably against fungal pathogens of the genus Phacopsora,
most preferably against Phakopsora pachyrhizi and Phakopsora
meibomiae, also known as soybean rust, in transgenic plants,
transgenic plant parts, or transgenic plant cells by overexpressing
one or more MybTF nucleic acids.
[0022] A further object is to provide transgenic plants resistant
against fungal pathogens, preferably rust pathogens (i.e., fungal
pathogens of the order Pucciniales), preferably of the family
Phacopsoraceae, more preferably against fungal pathogens of the
genus Phacopsora, most preferably against Phakopsora pachyrhizi and
Phakopsora meibomiae, also known as soybean rust, a method for
producing such plants as well as a vector construct useful for the
above methods.
[0023] Therefore, the present invention also refers to a
recombinant vector construct and a transgenic plant, transgenic
plant part, or transgenic plant cell comprising an exogenous MybTF
nucleic acid. Furthermore, a method for the production of a
transgenic plant, transgenic plant part or transgenic plant cell
using the nucleic acid of the present invention is claimed herein.
In addition, the use of a nucleic acid or the recombinant vector of
the present invention for the transformation of a plant, plant
part, or plant cell is claimed herein.
[0024] The objects of the present invention, as outlined above, are
achieved by the subject-matter of the main claims. Preferred
embodiments of the invention are defined by the subject matter of
the dependent claims.
BRIEF SUMMARY OF THE INVENTION
[0025] The object of the present invention is inter alia to provide
a method of increasing resistance against fungal pathogens,
preferably rust pathogens (i.e., fungal pathogens of the order
[0026] Pucciniales), preferably against fungal pathogens of the
family Phacopsoraceae, more preferably against fungal pathogens of
the genus Phacopsora, most preferably against Phakopsora pachyrhizi
and Phakopsora meibomiae, also known as soybean rust.
[0027] Surprisingly, we found that resistance against fungal
pathogens, in particular of the order Pucciniales, for example
soybean rust, can be enhanced by increasing the expression of a
MybTF protein.
[0028] The present invention therefore provides a method of
increasing resistance against fungal pathogens, preferably rust
pathogens (i.e., fungal pathogens of the order Pucciniales),
preferably against fungal pathogens of the family Phacopsoraceae,
more preferably against fungal pathogens of the genus Phacopsora,
most preferably against Phakopsora pachyrhizi and Phakopsora
meibomiae, also known as soybean rust, in transgenic plants,
transgenic plant parts, or transgenic plant cells by overexpressing
one or more MybTF nucleic acids.
[0029] A further object is to provide transgenic plants resistant
against fungal pathogens, preferably rust pathogens (i.e., fungal
pathogens of the order Pucciniales), preferably of the family
Phacopsoraceae, more preferably against fungal pathogens of the
genus Phacopsora, most preferably against Phakopsora pachyrhizi and
Phakopsora meibomiae, also known as soybean rust, a method for
producing such plants as well as a vector construct useful for the
above methods.
[0030] Therefore, the present invention also refers to a
recombinant vector construct and a transgenic plant, transgenic
plant part, or transgenic plant cell comprising an exogenous MybTF
nucleic acid. Furthermore, a method for the production of a
transgenic plant, transgenic plant part or transgenic plant cell
using the nucleic acid of the present invention is claimed herein.
In addition, the use of a nucleic acid or the recombinant vector of
the present invention for the transformation of a plant, plant
part, or plant cell is claimed herein.
[0031] The objects of the present invention, as outlined above, are
achieved by the subject-matter of the main claims. Preferred
embodiments of the invention are defined by the subject matter of
the dependent claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 shows the scoring system used to determine the level
of diseased leaf area of wildtype and transgenic soy plants against
the rust fungus P. pachyrhizi (as described in GODOY, C. V., KOGA,
L. J. & CANTERI, M. G. Diagrammatic scale for assessment of
soybean rust severity. Fitopatologia Brasileira 31:063-068.
2006).
[0033] FIG. 2 shows the schematic illustration of the plant
transformation vector harboring the fragment of MybTF DNA under
control of the parsley ubiquitine promoter.
[0034] FIG. 3 shows the alignment of the Arabidopsis MybTF genomic
sequence (AT3G29020-genomic, TAIR accession No 4010724011, SEQ ID
NO: 8), the two putative coding sequences of MybTF, which are
derived from the genomic sequence, MybTF CDS1 (At3G29020.1-CDS,
accession No NM.sub.--113823, SEQ-ID NO: 6) and MybTF CDS2
(At3G29020.2-CDS, TAIR accession No 4010715313, SEQ ID NO: 4) and
the sequence of the MybTF sequence as used in the examples
(MybTF-DNA, SEQ ID NO: 1).
[0035] FIG. 4 shows the result of the scoring of 15 transgenic soy
plants (To generation) expressing the MybTF overexpression vector
construct. To soybean plants harbouring the MybTF expression
cassette were inoculated with spores of Phakopsora pachyrhizi. The
expression of the MybTF was checked by RT-PCR. The evaluation of
the diseased leaf area on all leaves was performed 14 days after
inoculation. The average of the percentage of the leaf area showing
fungal colonies or strong yellowing/browning on all leaves was
considered as diseased leaf area. At all 15 soybean To plants
expressing the MybTF (expression checked by RT-PCR) were evaluated
in parallel to non-transgenic control plants. The average of the
diseased leaf area is shown in FIG. 4. Overexpression of MybTF
significantly (***: p<0.001) reduces the diseased leaf area in
comparison to non-transgenic control plants by 72.3%.
[0036] FIG. 5 contains a brief description of the sequences of the
sequence listing.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention may be understood more readily by
reference to the following detailed description of the preferred
embodiments of the invention and the examples included herein.
DEFINITIONS
[0038] Unless otherwise noted, the terms used herein are to be
understood according to conventional usage by those of ordinary
skill in the relevant art. In addition to the definitions of terms
provided herein, definitions of common terms in molecular biology
may also be found in Rieger et al., 1991 Glossary of genetics:
classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in
Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1998
Supplement).
[0039] It is to be understood that as used in the specification and
in the claims, "a" or "an" can mean one or more, depending upon the
context in which it is used. Thus, for example, reference to "a
cell" can mean that at least one cell can be utilized. It is to be
understood that the terminology used herein is for the purpose of
describing specific embodiments only and is not intended to be
limiting.
[0040] Throughout this application, various publications are
referenced. The disclosures of all of these publications and those
references cited within those publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the state of the art to which this invention
pertains. Standard techniques for cloning, DNA isolation,
amplification and purification, for enzymatic reactions involving
DNA ligase, DNA polymerase, restriction endonucleases and the like,
and various separation techniques are those known and commonly
employed by those skilled in the art. A number of standard
techniques are described in Sambrook et al., 1989 Molecular
Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview,
N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor
Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part
I; Wu (Ed.) 1979 Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth.
Enzymol. 100 and 101; Grossman and Moldave (Eds.) 1980 Meth.
Enzymol. 65; Miller (Ed.) 1972 Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and
Primrose, 1981 Principles of Gene Manipulation, University of
California Press, Berkeley; Schleif and Wensink, 1982 Practical
Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I
and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985
Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and
Hollaender 1979 Genetic Engineering: Principles and Methods, Vols.
1-4, Plenum Press, New York. Abbreviations and nomenclature, where
employed, are deemed standard in the field and commonly used in
professional journals such as those cited herein.
[0041] "Homologues" of a protein encompass peptides, oligopeptides,
polypeptides, proteins and/or enzymes having amino acid
substitutions, deletions and/or insertions relative to the
unmodified protein in question and having similar functional
activity as the unmodified protein from which they are derived.
[0042] "Homologues" of a nucleic acid encompass nucleotides and/or
polynucleotides having nucleic acid substitutions, deletions and/or
insertions relative to the unmodified nucleic acid in question,
wherein the protein coded by such nucleic acids has similar
functional activity as the unmodified protein coded by the
unmodified nucleic acid from which they are derived. In particular,
homologues of a nucleic acid may encompass substitutions on the
basis of the degenerative amino acid code.
[0043] The terms "identity", "homology" and "similarity" are used
herein interchangeably. "Identity" or "homology" or "similarity"
between two nucleic acids sequences or amino acid sequences refers
in each case over the entire length of the respective MybTF nucleic
acid sequence or MybTF amino acid sequence.
[0044] Preferably, "percentage of sequence identity" is calculated
by comparing two optimally aligned sequences over a particular
region, determining the number of positions at which the identical
base or amino acid occurs in both sequences in order to yield the
number of matched positions, dividing the number of such positions
by the total number of positions in the region being compared and
multiplying the result by 100.
[0045] Methods for the alignment of sequences for comparison are
well known in the art, such methods include GAP, BESTFIT, BLAST,
FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch
((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning
the complete sequences) alignment of two sequences that maximizes
the number of matches and minimizes the number of gaps. The BLAST
algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10)
calculates percent sequence identity or similarity or homology and
performs a statistical analysis of the identity or similarity or
homology between the two sequences. The software for performing
BLAST analysis is publicly available through the National Centre
for Biotechnology Information (NCBI). Homologues may readily be
identified using, for example, the ClustalW multiple sequence
alignment algorithm (version 1.83), with the default pairwise
alignment parameters, and a scoring method in percentage. Global
percentages of similarity/homology/identity may also be determined
using one of the methods available in the MatGAT software package
(Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT:
an application that generates similarity/homology/identity matrices
using protein or DNA sequences.). Minor manual editing may be
performed to optimise alignment between conserved motifs, as would
be apparent to a person skilled in the art. Furthermore, instead of
using full-length sequences for the identification of homologues,
specific domains may also be used. The sequence identity values may
be determined over the entire nucleic acid or amino acid sequence
or over selected domains or conserved motif(s), using the programs
mentioned above using the default parameters. For local alignments,
the SmithWaterman algorithm is particularly useful (Smith T F,
Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
[0046] The sequence identity may also be calculated by means of the
Vector NTI Suite 7.1 program of the company Informax (USA)
employing the Clustal Method (Higgins D G, Sharp P M. Fast and
sensitive multiple sequence alignments on a microcomputer. Comput
Appl. Biosci. 1989 April; 5(2):151-1) with the following
settings:
[0047] Multiple Alignment Parameter:
TABLE-US-00001 Gap opening penalty 10 Gap extension penalty 10 Gap
separation penalty range 8 Gap separation penalty off % identity
for alignment delay 40 Residue specific gaps off Hydrophilic
residue gap off Transition weighing 0
[0048] Pairwise Alignment Parameter:
TABLE-US-00002 FAST algorithm on K-tuple size 1 Gap penalty 3
Window size 5 Number of best diagonals 5
[0049] Alternatively the identity may be determined according to
Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo,
Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple
sequence alignment with the Clustal series of programs. (2003)
Nucleic Acids Res 31 (13):3497-500, the web page:
http://www.ebi.ac.uk/Tools/clustalw/index.html# and the following
settings
TABLE-US-00003 DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty
6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein Gap
Extension Penalty 0.2 Protein matrix Gonnet Protein/DNA ENDGAP -1
Protein/DNA GAPDIST 4
[0050] Sequence identity between the nucleic acid or protein useful
according to the present invention and the MybTF nucleic acids or
MybTF proteins may be optimized by sequence comparison and
alignment algorithms known in the art (see Gribskov and Devereux,
Sequence Analysis Primer, Stockton Press, 1991, and references
cited therein) and calculating the percent difference between the
nucleotide or protein sequences by, for example, the SmithWaterman
algorithm as implemented in the BESTFIT software program using
default parameters (e.g., University of Wisconsin Genetic Computing
Group).
[0051] A "deletion" refers to removal of one or more amino acids
from a protein or to the removal of one or more nucleic acids from
DNA, ssRNA and/or dsRNA.
[0052] An "insertion" refers to one or more amino acid residues or
nucleic acid residues being introduced into a predetermined site in
a protein or the nucleic acid.
[0053] A "substitution" refers to replacement of amino acids of the
protein with other amino acids having similar properties (such as
similar hydrophobicity, hydrophilicity, antigenicity, propensity to
form or break .alpha.-helical structures or beta-sheet
structures).
[0054] On the nucleic acid level a substitution refers to a
replacement of one or more nucleotides with other nucleotides
within a nucleic acid, wherein the protein coded by the modified
nucleic acid has a similar function. In particular homologues of a
nucleic acid encompass substitutions on the basis of the
degenerative amino acid code.
[0055] Amino acid substitutions are typically of single residues,
but may be clustered depending upon functional constraints placed
upon the protein and may range from 1 to 10 amino acids; insertions
or deletion will usually be of the order of about 1 to 10 amino
acid residues. The amino acid substitutions are preferably
conservative amino acid substitutions. Conservative substitution
tables are well known in the art (see for example Taylor W. R.
(1986) The classification of amino acid conservation J Theor Biol.,
119:205-18 and Table 1 below).
TABLE-US-00004 TABLE 1 Examples of conserved amino acid
substitutions Conservative Conservative Residue Substitutions
Residue Substitutions A G, V, I, L, M L M, I, V, A, G C S, T N Q E
D Q N D E P G A, V, I, L, M S T, C F Y, W R K, H I V, A, G, L, M T
S, C H R, K W Y, F K R, H V I, A, G, L, M M L, I, V, A, G Y F,
W
[0056] Amino acid substitutions, deletions and/or insertions may
readily be made using peptide synthetic techniques well known in
the art, such as solid phase peptide synthesis and the like, or by
recombinant DNA manipulation.
[0057] Methods for the manipulation of DNA sequences to produce
substitution, insertion or deletion variants of a protein are well
known in the art. For example, techniques for making substitution
mutations at predetermined sites in DNA are well known to those
skilled in the art and include M13 mutagenesis, T7-Gene in vitro
mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed
mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated
site-directed mutagenesis or other site-directed mutagenesis
protocols.
[0058] Orthologues and paralogues encompass evolutionary concepts
used to describe the ancestral relationships of genes. Paralogues
are genes within the same species that have originated through
duplication of an ancestral gene; orthologues are genes from
different organisms that have originated through speciation, and
are also derived from a common ancestral gene.
[0059] The terms "encode" or "coding for" is used for the
capability of a nucleic acid to contain the information for the
amino acid sequence of a protein via the genetic code, i.e., the
succession of codons each being a sequence of three nucleotides,
which specify which amino acid will be added next during protein
synthesis. The terms "encode" or "coding for" therefore includes
all possible reading frames of a nucleic acid. Furthermore, the
terms "encode" or "coding for" also applies to a nucleic acid,
which coding sequence is interrupted by noncoding nucleic acid
sequences, which are removed prior translation, e.g., a nucleic
acid sequence comprising introns.
[0060] The term "domain" refers to a set of amino acids conserved
at specific positions along an alignment of sequences of
evolutionarily related proteins. While amino acids at other
positions can vary between homologues, amino acids that are highly
conserved at specific positions indicate amino acids that are
likely essential in the structure, stability or function of a
protein.
[0061] Specialist databases exist for the identification of
domains, for example, SMART (Schultz et al. (1998) Proc. Natl.
Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids
Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res.
31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized
profile syntax for biomolecular sequences motifs and its function
in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd
International Conference on Intelligent Systems for Molecular
Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D.,
Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids.
Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids
Research 30(1): 276-280 (2002)). A set of tools for in sflico
analysis of protein sequences is available on the ExPASy proteomics
server (Swiss Institute of Bioinformatics (Gasteiger et al.,
ExPASy: the proteomics server for in-depth protein knowledge and
analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs
may also be identified using routine techniques, such as by
sequence alignment.
[0062] As used herein the terms "fungal-resistance", "resistant to
a fungus" and/or "fungal-resistant" mean reducing, preventing, or
delaying an infection by fungi. The term "resistance" refers to
fungal resistance. Resistance does not imply that the plant
necessarily has 100% resistance to infection. In preferred
embodiments, enhancing or increasing fungal resistance means that
resistance in a resistant plant is greater than 10%, greater than
20%, greater than 30%, greater than 40%, greater than 50%, greater
than 60%, greater than 70%, greater than 80%, greater than 90%, or
greater than 95% in comparison to a wild type plant.
[0063] As used herein the terms "soybean rust-resistance",
"resistant to a soybean rust", "soybean rust-resistant",
"rust-resistance", "resistant to a rust", or "rust-resistant" mean
reducing or preventing or delaying an infection of a plant, plant
part, or plant cell by Phacopsoracea, in particular Phakopsora
pachyrhizi and Phakopsora meibomiae--also known as soybean rust or
Asian Soybean Rust (ASR), as compared to a wild type plant, wild
type plant part, or wild type plant cell. Resistance does not imply
that the plant necessarily has 100% resistance to infection. In
preferred embodiments, enhancing or increasing rust resistance
means that rust resistance in a resistant plant is greater than
10%, greater than 20%, greater than 30%, greater than 40%, greater
than 50%, greater than 60%, greater than 70%, greater than 80%,
greater than 90%, or greater than 95% in comparison to a wild type
plant that is not resistant to soybean rust. Preferably the wild
type plant is a plant of a similar, more preferably identical,
genotype as the plant having increased resistance to the soybean
rust, but does not comprise an exogenous MybTF nucleic acid,
functional fragments thereof and/or an exogenous nucleic acid
capable of hybridizing with a MybTF nucleic acid.
[0064] The level of fungal resistance of a plant can be determined
in various ways, e.g. by scoring/measuring the infected leaf area
in relation to the overall leaf area. Another possibility to
determine the level of resistance is to count the number of soybean
rust colonies on the plant or to measure the amount of spores
produced by these colonies. Another way to resolve the degree of
fungal infestation is to specifically measure the amount of rust
DNA by quantitative (q) PCR. Specific probes and primer sequences
for most fungal pathogens are available in the literature
(Frederick R D, Snyder C L, Peterson G L, et al. 2002 Polymerase
chain reaction assays for the detection and discrimination of the
rust pathogens Phakopsora pachyrhizi and P. meibomiae,
Phytopathology 92(2) 217-227).
[0065] The term "hybridization" as used herein includes "any
process by which a strand of nucleic acid molecule joins with a
complementary strand through base pairing" (J. Coombs (1994)
Dictionary of Biotechnology, Stockton Press, New York).
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acid molecules) is impacted
by such factors as the degree of complementarity between the
nucleic acid molecules, stringency of the conditions involved, the
Tm of the formed hybrid, and the G:C ratio within the nucleic acid
molecules.
[0066] As used herein, the term "Tm" is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. The equation for
calculating the Tm of nucleic acid molecules is well known in the
art. As indicated by standard references, a simple estimate of the
Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C),
when a nucleic acid molecule is in aqueous solution at 1 M NaCl
(see e.g., Anderson and Young, Quantitative Filter Hybridization,
in Nucleic Acid Hybridization (1985). Other references include more
sophisticated computations, which take structural as well as
sequence characteristics into account for the calculation of Tm.
Stringent conditions, are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6.
[0067] In particular, the term "stringency conditions" refers to
conditions, wherein 100 contigous nucleotides or more, 150
contigous nucleotides or more, 200 contigous nucleotides or more or
250 contigous nucleotides or more which are a fragment or identical
to the complementary nucleic acid molecule (DNA, RNA, ssDNA or
ssRNA) hybridizes under conditions equivalent to hybridization in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C. or 65.degree. C., preferably at 65.degree. C., with a specific
nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the
hybridizing conditions are equivalent to hybridization in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50.degree. C. with
washing in 1.times.SSC, 0.1% SDS at 50.degree. C. or 65.degree. C.,
preferably 65.degree. C., more preferably the hybridizing
conditions are equivalent to hybridization in 7% sodium dodecyl
sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50.degree. C. with washing
in 0.1.times.SSC, 0.1% SDS at 50.degree. C. or 65.degree. C.,
preferably 65.degree. C. Preferably, the complementary nucleotides
hybridize with a fragment or the whole MybTF nucleic acids.
Alternatively, preferred hybridization conditions encompass
hybridisation at 65.degree. C. in 1.times.SSC or at 42.degree. C.
in 1.times.SSC and 50% formamide, followed by washing at 65.degree.
C. in 0.3.times.SSC or hybridisation at 50.degree. C. in
4.times.SSC or at 40.degree. C. in 6.times.SSC and 50% formamide,
followed by washing at 50.degree. C. in 2.times.SSC. Further
preferred hybridization conditions are 0.1% SDS, 0.1 SSD and
65.degree. C.
[0068] The term "plant" is intended to encompass plants at any
stage of maturity or development, as well as any tissues or organs
(plant parts) taken or derived from any such plant unless otherwise
clearly indicated by context. Plant parts include, but are not
limited to, plant cells, stems, roots, flowers, ovules, stamens,
seeds, leaves, embryos, meristematic regions, callus tissue, anther
cultures, gametophytes, sporophytes, pollen, microspores,
protoplasts, hairy root cultures, and/or the like. The present
invention also includes seeds produced by the plants of the present
invention. Preferably, the seeds comprise the exogenous MybTF
nucleic acids. In one embodiment, the seeds can develop into plants
with increased resistance to fungal infection as compared to a
wild-type variety of the plant seed. As used herein, a "plant cell"
includes, but is not limited to, a protoplast, gamete producing
cell, and a cell that regenerates into a whole plant. Tissue
culture of various tissues of plants and regeneration of plants
therefrom is well known in the art and is widely published.
[0069] Reference herein to an "endogenous" nucleic acid and/or
protein refers to the nucleic acid and/or protein in question as
found in a plant in its natural form (i.e., without there being any
human intervention).
[0070] The term "exogenous" nucleic acid refers to a nucleic acid
that has been introduced in a plant by means of genetechnology. An
"exogenous" nucleic acid can either not occur in a plant in its
natural form, be different from the nucleic acid in question as
found in a plant in its natural form, or can be identical to a
nucleic acid found in a plant in its natural form, but integrated
not within their natural genetic environment. The corresponding
meaning of "exogenous" is applied in the context of protein
expression. For example, a transgenic plant containing a transgene,
i.e., an exogenous nucleic acid, may, when compared to the
expression of the endogenous gene, encounter a substantial increase
of the expression of the respective gene or protein in total. A
transgenic plant according to the present invention includes an
exogenous MybTF nucleic acid integrated at any genetic loci and
optionally the plant may also include the endogenous gene within
the natural genetic background.
[0071] For the purposes of the invention, "recombinant" means with
regard to, for example, a nucleic acid sequence, a nucleic acid
molecule, an expression cassette or a vector construct comprising
any one or more MybTF nucleic acids, all those constructions
brought about by man by genetechnological methods in which either
[0072] (a) the sequences of the MybTF nucleic acids or a part
thereof, or [0073] (b) genetic control sequence(s) which is
operably linked with the MybTF nucleic acid sequence according to
the invention, for example a promoter, or [0074] (c) a) and b) are
not located in their natural genetic environment or have been
modified by man by genetechnological methods. The modification may
take the form of, for example, a substitution, addition, deletion,
inversion or insertion of one or more nucleotide residues. The
natural genetic environment is understood as meaning the natural
genomic or chromosomal locus in the original plant or the presence
in a genomic library or the combination with the natural
promoter.
[0075] For instance, a naturally occurring expression cassette--for
example the naturally occurring combination of the natural promoter
of the nucleic acid sequences with the corresponding nucleic acid
sequence encoding a protein useful in the methods of the present
invention, as defined above--becomes a recombinant expression
cassette when this expression cassette is modified by man by
non-natural, synthetic ("artificial") methods such as, for example,
mutagenic treatment. Suitable methods are described, for example,
in U.S. Pat. No. 5,565,350, WO 00/15815 or US200405323.
Furthermore, a naturally occurring expression cassette--for example
the naturally occurring combination of the natural promoter of the
nucleic acid sequences with the corresponding nucleic acid sequence
encoding a protein useful in the methods of the present invention,
as defined above--becomes a recombinant expression cassette when
this expression cassette is not integrated in the natural genetic
environment but in a different genetic environment.
[0076] The term "isolated nucleic acid" or "isolated protein"
refers to a nucleic acid or protein that is not located in its
natural environment, in particular its natural cellular
environment. Thus, an isolated nucleic acid or isolated protein is
essentially separated from other components of its natural
environment. However, the skilled person in the art is aware that
preparations of an isolated nucleic acid or an isolated protein can
display a certain degree of impurity depending on the isolation
procedure used. Methods for purifying nucleic acids and proteins
are well known in the art. The isolated gene may be isolated from
an organism or may be manmade, for example by chemical synthesis.
In this regard, a recombinant nucleic acid may also be in an
isolated form.
[0077] As used herein, the term "transgenic" refers to an organism,
e.g., a plant, plant cell, callus, plant tissue, or plant part that
exogenously contains the nucleic acid, recombinant construct,
vector or expression cassette described herein or a part thereof
which is preferably introduced by non-essentially biological
processes, preferably by Agrobacteria transformation. The
recombinant construct or a part thereof is stably integrated into a
chromosome, so that it is passed on to successive generations by
clonal propagation, vegetative propagation or sexual propagation.
Preferred successive generations are transgenic too. Essentially
biological processes may be crossing of plants and/or natural
recombination.
[0078] A transgenic plant, plants cell or tissue for the purposes
of the invention is thus understood as meaning that an exogenous
MybTF nucleic acid, recombinant construct, vector or expression
cassette including one or more MybTF nucleic acids is integrated
into the genome by means of genetechnology.
[0079] A "wild type" plant, "wild type" plant part, or "wild type"
plant cell means that said plant, plant part, or plant cell does
not express exogenous MybTF nucleic acid or exogenous MybTF
protein.
[0080] Natural locus means the location on a specific chromosome,
preferably the location between certain genes, more preferably the
same sequence background as in the original plant which is
transformed.
[0081] Preferably, the transgenic plant, plant cell or tissue
thereof expresses the MybTF nucleic acids, MybTF constructs or
MybTF expression cassettes described herein.
[0082] The term "expression" or "gene expression" means the
transcription of a specific gene or specific genes or specific
genetic vector construct. The term "expression" or "gene
expression" in particular means the transcription of a gene or
genes or genetic vector construct into structural RNA (rRNA, tRNA),
or mRNA with or without subsequent translation of the latter into a
protein. The process includes transcription of DNA and processing
of the resulting RNA product. The term "expression" or "gene
expression" can also include the translation of the mRNA and
therewith the synthesis of the encoded protein, i.e., protein
expression.
[0083] The term "increased expression" or "enhanced expression" or
"overexpression" or "increase of content" as used herein means any
form of expression that is additional to the original wild-type
expression level. For the purposes of this invention, the original
wild-type expression level might also be zero (absence of
expression).
[0084] Methods for increasing expression of genes or gene products
are well documented in the art and include, for example,
overexpression driven by appropriate promoters, the use of
transcription enhancers or translation enhancers. Isolated nucleic
acids which serve as promoter or enhancer elements may be
introduced in an appropriate position (typically upstream) of a
non-heterologous form of a polynucleotide so as to upregulate
expression of a nucleic acid encoding the protein of interest. For
example, endogenous promoters may be altered in vivo by mutation,
deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350;
Zarling et al., WO9322443), or isolated promoters may be introduced
into a plant cell in the proper orientation and distance from a
gene of the present invention so as to control the expression of
the gene.
[0085] The term "functional fragment" refers to any nucleic acid or
protein which comprises merely a part of the fulllength nucleic
acid or fulllength protein, respectively, but still provides the
same function, e.g., fungal resistance, when expressed or repressed
in a plant, respectively. Preferably, the fragment comprises at
least 70%, at least 80%, at least 90% at least 95%, at least 98%,
at least 99% of the original sequence. Preferably, the functional
fragment comprises contiguous nucleic acids or amino acids as in
the original nucleic acid or original protein, respectively. In one
embodiment the fragment of any of the MybTF nucleic acids has an
identity as defined above over a length of at least 70%, at least
75%, at least 90% of the nucleotides of the respective MybTF
nucleic acid.
[0086] The term "splice variant" as used herein encompasses
variants of a nucleic acid sequence in which selected introns
and/or exons or parts thereof have been excised, replaced,
displaced or added, or in which introns have been shortened or
lengthened. Thus, a splice variant can have one or more or even all
introns removed or added or partially removed or partially added.
According to this definition, a cDNA is considered as a splice
variant of the respective intron-containing genomic sequence and
vice versa. Such splice variants may be found in nature or may be
manmade. Methods for predicting and isolating such splice variants
are well known in the art (see for example Foissac and Schiex
(2005) BMC Bioinformatics 6: 25).
[0087] In cases where overexpression of nucleic acid is desired,
the term "similar functional activity" or "similar function" means
that any homologue and/or fragment provide fungal resistance when
expressed in a plant. Preferably similar functional activity means
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least 98%, at least 99% or 100% or higher
fungal resistance compared with functional activity provided by the
exogenous expression of the MybTF nucleotide sequence as defined by
SEQ ID NO: 6, 4, 2, 3, or 1.
[0088] The term "increased activity" or "enhanced activity" as used
herein means any protein having increased activity and which
provides an increased fungal resistance compared with the wildtype
plant merely expressing the respective endogenous MybTF nucleic
acid. As far as overexpression is concerned, for the purposes of
this invention, the original wild-type expression level might also
be zero (absence of expression).
[0089] With respect to a vector construct and/or the recombinant
nucleic acid molecules, the term "operatively linked" is intended
to mean that the nucleic acid to be expressed is linked to the
regulatory sequence, including promoters, terminators, enhancers
and/or other expression control elements (e.g., polyadenylation
signals), in a manner which allows for expression of the nucleic
acid (e.g., in a host plant cell when the vector is introduced into
the host plant cell). Such regulatory sequences are described, for
example, in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber
and Crosby, in: Methods in Plant Molecular Biology and
Biotechnology, Eds. Glick and Thompson, Chapter 7, 89-108, CRC
Press: Boca Raton, Fla., including the references therein.
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cells and
those that direct expression of the nucleotide sequence only in
certain host cells or under certain conditions. It will be
appreciated by those skilled in the art that the design of the
vector can depend on such factors as the choice of the host cell to
be transformed, the level of expression of nucleic acid desired,
and the like.
[0090] The term "introduction" or "transformation" as referred to
herein encompass the transfer of an exogenous polynucleotide into a
host cell, irrespective of the method used for transfer. Plant
tissue capable of subsequent clonal propagation, whether by
organogenesis or embryogenesis, may be transformed with a vector
construct of the present invention and a whole plant regenerated
there from. The particular tissue chosen will vary depending on the
clonal propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue
(e.g., apical meristem, axillary buds, and root meristems), and
induced meristem tissue (e.g., cotyledon meristem and hypocotyl
meristem). The polynucleotide may be transiently or stably
introduced into a host cell and may be maintained non-integrated,
for example, as a plasmid. Alternatively, it may be integrated into
the host genome. The host genome includes the nucleic acid
contained in the nucleus as well as the nucleic acid contained in
the plastids, e.g., chloroplasts, and/or mitochondria. The
resulting transformed plant cell may then be used to regenerate a
transformed plant in a manner known to persons skilled in the
art.
[0091] The term "terminator" encompasses a control sequence which
is a DNA sequence at the end of a transcriptional unit which
signals 3' processing and polyadenylation of a primary transcript
and termination of transcription. The terminator can be derived
from the natural gene, from a variety of other plant genes, or from
T-DNA. The terminator to be added may be derived from, for example,
the nopaline synthase or octopine synthase genes, or alternatively
from another plant gene, or less preferably from any other
eukaryotic gene.
DETAILED DESCRIPTION
MybTF Nucleic Acids
[0092] The MybTF nucleic acid to be overexpressed in order to
achieve increased resistance to fungal pathogens, e.g., of the
family Phacopsoraceae, for example soybean rust, is preferably a
nucleic acid coding for a MybTF protein, preferably of the R2R3-MYB
family, and is preferably as defined by SEQ ID NO: 2, 3, 1, 6, 4,
8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, or 55, or a fragment, homolog, derivative, orthologue or
paralogue thereof, or a splice variant thereof. Preferably, the
nucleic acid coding for a MybTF protein of the present invention
has at least 70% sequence identity, at least 80%, at least 90%, at
least 92%, at least 95%, at least 97%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55, or is a functional fragment thereof, or a
splice variant thereof. Most preferred is at least 90% identity, at
least 92%, at least 95%, at least 97%, more preferred is at least
98% or at least 99% identity with SEQ ID NO: 2, 3, 1, 6, 4, 8,
9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
or 55.
[0093] Preferably, the MybTF nucleic acid to be overexpressed in
order to achieve increased resistance to fungal pathogens, e.g., of
the family Phacopsoraceae, for example soybean rust, is preferably
a nucleic acid coding for a MybTF protein, and is preferably as
defined by SEQ ID NO: 2, or a fragment, homolog, derivative,
orthologue or paralogue thereof, or a splice variant thereof.
Preferably, the nucleic acid coding for a MybTF protein of the
present invention has at least 70% sequence identity, at least 80%,
at least 90%, at least 92%, at least 95%, at least 97%, at least
98%, at least 99% sequence identity, or even 100% sequence identity
with SEQ ID NO: 2 or is a functional fragment thereof, or a splice
variant thereof. Most preferred is at least 90% identity, at least
92%, at least 95%, at least 97% identity, more preferred is at
least 98% or at least 99% identity with SEQ ID NO: 2.
[0094] More preferably, the MybTF nucleic acid to be overexpressed
in order to achieve increased resistance to fungal pathogens, e.g.,
of the family Phacopsoraceae, for example soybean rust, is
preferably a nucleic acid coding for a MybTF protein, and is
preferably as defined by SEQ ID NO: 1, or a fragment, homolog,
derivative, orthologue or paralogue thereof, or a splice variant
thereof. Preferably, the nucleic acid coding for a MybTF protein of
the present invention has at least 70% sequence identity, at least
80%, at least 90%, at least 92%, at least 95%, at least 97%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 1 or is a functional fragment thereof, or
a splice variant thereof. Most preferred is at least 92%, at least
95%, at least 97% identity, more preferred is at least 98% or at
least 99% identity with SEQ ID NO: 1.
[0095] Preferably, the MybTF nucleic acid to be overexpressed in
order to achieve increased resistance to fungal pathogens, e.g., of
the family Phacopsoraceae, for example soybean rust, is preferably
a nucleic acid coding for a MybTF protein, and is preferably as
defined by SEQ ID NO: 3, or a fragment, homolog, derivative,
orthologue or paralogue thereof, or a splice variant thereof.
Preferably, the nucleic acid coding for a MybTF protein of the
present invention has at least 70% sequence identity, at least 80%,
at least 90%, at least 92%, at least 95%, at least 97%, at least
98%, at least 99% sequence identity, or even 100% sequence identity
with SEQ ID NO: 3 or is a functional fragment thereof, or a splice
variant thereof. Most preferred is at least 90% identity, at least
92%, at least 95%, at least 97% identity, more preferred is at
least 98% or at least 99% identity with SEQ ID NO: 3.
[0096] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule consisting of or comprising a nucleic acid selected
from the group consisting of: [0097] (i) a nucleic acid having in
increasing order of preference at least 70%, at least 71%, at least
72%, at least 73%, at least 74%, at least 75%, at least 76%, at
least 77%, at least 78%, at least 79%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the nucleic acid sequence represented by SEQ
ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, or 55, or a functional fragment,
derivative, orthologue, or paralogue thereof, or a splice variant
thereof; [0098] (ii) a nucleic acid encoding a MybTF protein
comprising an amino acid sequence having in increasing order of
preference at least 70%, at least 71%, at least 72%, at least 73%,
at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
the amino acid sequence represented by SEQ ID NO: 7, 5, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, or a
functional fragment, derivative, orthologue, or paralogue thereof;
preferably the MybTF protein has essentially the same biological
activity as a MybTF protein encoded by SEQ ID NO: 2, 3, 1, 6, or 4;
preferably the MybTF protein confers enhanced fungal resistance
relative to control plants; [0099] (iii) a nucleic acid molecule
which hybridizes with a complementary sequence of any of the
nucleic acid molecules of (i) or (ii) under high stringency
hybridization conditions; preferably encoding a MybTF protein;
preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 5 or 7; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0100] (iv) a nucleic acid encoding the same MybTF
protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0101] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule consisting of or comprising a nucleic acid selected
from the group consisting of: [0102] (i) a nucleic acid having in
increasing order of preference at least 70%, at least 71%, at least
72%, at least 73%, at least 74%, at least 75%, at least 76%, at
least 77%, at least 78%, at least 79%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the nucleic acid sequence represented by SEQ
ID NO: 1 or 8, or a functional fragment, derivative, orthologue, or
paralogue thereof, or a splice variant thereof; [0103] (ii) a
nucleic acid encoding a MybTF protein having in increasing order of
preference at least 70%, at least 71%, at least 72%, at least 73%,
at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
the amino acid sequence represented by SEQ ID NO: 7 or 5, or a
functional fragment, derivative, orthologue, or paralogue thereof;
preferably the MybTF protein has essentially the same biological
activity as a MybTF protein encoded by SEQ ID NO: 1 or 8,
preferably the MybTF protein confers enhanced fungal resistance
relative to control plants; [0104] (iii) a nucleic acid molecule
which hybridizes with a complementary sequence of any of the
nucleic acid molecules of (i) or (ii) under high stringency
hybridization conditions; preferably encoding a MybTF protein;
preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0105] (iv) a nucleic acid encoding the same MybTF
protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0106] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule comprising a nucleic acid selected from the group
consisting of: [0107] (i) a nucleic acid having in increasing order
of preference at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% sequence identity
to the nucleic acid sequence represented by SEQ ID NO: 2 or 3, or a
functional fragment, derivative, orthologue, or paralogue thereof,
or a splice variant thereof; [0108] (ii) a nucleic acid encoding a
MybTF protein having in increasing order of preference at least
70%, at least 71%, at least 72%, at least 73%, at least 74%, at
least 75%, at least 76%, at least 77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or 100% sequence identity to the amino acid
sequence represented by SEQ ID NO: 7 or 5, or a functional
fragment, derivative, orthologue, or paralogue thereof; preferably
the MybTF protein has essentially the same biological activity as a
MybTF protein encoded by SEQ ID NO: 2 or 3, preferably the MybTF
protein confers enhanced fungal resistance relative to control
plants; [0109] (iii) a nucleic acid molecule which hybridizes with
a complementary sequence of any of the nucleic acid molecules of
(i) or (ii) under high stringency hybridization conditions;
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; and [0110] (iv) a nucleic
acid encoding the same MybTF protein as the MybTF nucleic acids of
(i) to (iii) above, but differing from the MybTF nucleic acids of
(i) to (iii) above due to the degeneracy of the genetic code.
[0111] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule comprising a nucleic acid selected from the group
consisting of: [0112] (i) a nucleic acid having in increasing order
of preference at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% sequence identity
to the nucleic acid sequence represented by SEQ ID NO: 2 or 6, or a
functional fragment, derivative, orthologue, or paralogue thereof,
or a splice variant thereof; [0113] (ii) a nucleic acid encoding a
MybTF protein having in increasing order of preference at least
70%, at least 71%, at least 72%, at least 73%, at least 74%, at
least 75%, at least 76%, at least 77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or 100% sequence identity to the amino acid
sequence represented by SEQ ID NO: 7, or a functional fragment,
derivative, orthologue, or paralogue thereof; preferably the MybTF
protein has essentially the same biological activity as a MybTF
protein encoded by SEQ ID NO: 2 or 6, preferably the MybTF protein
confers enhanced fungal resistance relative to control plants;
[0114] (iii) a nucleic acid molecule which hybridizes with a
complementary sequence of any of the nucleic acid molecules of (i)
or (ii) under high stringency hybridization conditions; preferably
encoding a MybTF protein; preferably wherein the nucleic acid
molecule codes for a polypeptide which has essentially identical
properties to the polypeptide described in SEQ ID NO: 7; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; and [0115] (iv) a nucleic acid encoding the same
MybTF protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0116] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule comprising a nucleic acid selected from the group
consisting of: [0117] (i) a nucleic acid having in increasing order
of preference at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% sequence identity
to the nucleic acid sequence represented by SEQ ID NO: 3 or 4, or a
functional fragment, derivative, orthologue, or paralogue thereof,
or a splice variant thereof; [0118] (ii) a nucleic acid encoding a
MybTF protein having in increasing order of preference at least
70%, at least 71%, at least 72%, at least 73%, at least 74%, at
least 75%, at least 76%, at least 77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or 100% sequence identity to the amino acid
sequence represented by SEQ ID NO: 5, or a functional fragment,
derivative, orthologue, or paralogue thereof; preferably the MybTF
protein has essentially the same biological activity as a MybTF
protein encoded by SEQ ID NO: 3 or 4, preferably the MybTF protein
confers enhanced fungal resistance relative to control plants;
[0119] (iii) a nucleic acid molecule which hybridizes with a
complementary sequence of any of the nucleic acid molecules of (i)
or (ii) under high stringency hybridization conditions; preferably
encoding a MybTF protein; preferably wherein the nucleic acid
molecule codes for a polypeptide which has essentially identical
properties to the polypeptide described in SEQ ID NO: 5; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; and [0120] (iv) a nucleic acid encoding the same
MybTF protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0121] Percentages of identity of a nucleic acid are indicated with
reference to the entire nucleotide region given in a sequence
specifically disclosed herein.
[0122] Preferably the portion of the MybTF nucleic acid is about
500-600, about 600-700, about 800-900, about 900-1000, about
1000-1100, about 1100-1200, about 1200-1300, or about 1300-1340
nucleotides, preferably consecutive nucleotides, preferably counted
from the 5' or 3' end of the nucleic acid, in length, of the
nucleic acid sequences given in SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or
55.
[0123] Preferably, the MybTF nucleic acid comprises at least about
500, at least about 600, at least about 700, at least about 800, at
least about 900, at least about 1100, at least about 1200, or at
least about 1300 nucleotides, preferably continuous nucleotides,
preferably counted from the 5' or 3' end of the nucleic acid or up
to the full length of the nucleic acid sequence set out in SEQ ID
NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, or 55.
[0124] Preferably, the MybTF nucleic acid comprises at least about
500, at least about 550, at least about 600, at least about 650, at
least about 700, at least about 750, at least about 800, at least
about 850, or at least about 900 nucleotides, preferably continuous
nucleotides, preferably counted from the 5' or 3' end of the
nucleic acid or up to the full length of the nucleic acid sequence
set out in SEQ ID NO: 2, 3, 1, 6, 4, 9-24, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, or 55.
[0125] Preferably the portion of the MybTF nucleic acid is about
500-550, about 550-600, about 600-650, about 650-700, about
675-708, about 700-750, about 750-800, about 800-850, about
850-900, or about 900-918 nucleotides, preferably consecutive
nucleotides, preferably counted from the 5' or 3' end of the
nucleic acid, in length, of the nucleic acid sequences given in SEQ
ID NO: 2, 3, 1, 6, 4, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, or 55.
[0126] Preferably, the MybTF nucleic acid comprises at least about
500, at least about 550, at least about 600, at least about 650, at
least about 700, at least about 750, at least about 800, at least
about 850, or at least about 900 nucleotides, preferably continuous
nucleotides, preferably counted from the 5' or 3' end of the
nucleic acid or up to the full length of the nucleic acid sequence
set out in SEQ ID NO: 2 or 3.
[0127] Preferably the portion of the MybTF nucleic acid is about
500-550, about 550-600, about 600-650, about 650-700, about
675-708, about 700-750, about 750-800, about 800-850, about
850-900, or about 900-918 nucleotides, preferably consecutive
nucleotides, preferably counted from the 5' or 3' end of the
nucleic acid, in length, of the nucleic acid sequences given in SEQ
ID NO: 2 or 3.
[0128] Preferably, the MybTF nucleic acid is a MybTF nucleic acid
splice variant. Preferred splice variants are splice variants of a
nucleic acid represented by SEQ ID NO: 1 or 8. Preferred MybTF
nucleic acids being a splice variant of SEQ ID NO: 1 or 8 are shown
in FIG. 3.
[0129] Preferably, the MybTF nucleic acid is an isolated nucleic
acid molecule comprising a splice variant of SEQ ID NO: 1 or 8,
wherein the splice variant is selected from the group consisting
of: [0130] (i) a nucleic acid having in increasing order of
preference at least 70%, at least 71%, at least 72%, at least 73%,
at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to
the nucleic acid sequence represented by SEQ ID NO: 4 or 6, or a
functional fragment, derivative, orthologue, or paralogue thereof;
[0131] (ii) a nucleic acid encoding a MybTF protein having in
increasing order of preference at least 70%, at least 71%, at least
72%, at least 73%, at least 74%, at least 75%, at least 76%, at
least 77%, at least 78%, at least 79%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the amino acid sequence represented by SEQ ID
NO: 7 or 5, or a functional fragment, derivative, orthologue, or
paralogue thereof; preferably the MybTF protein has essentially the
same biological activity as a MybTF protein encoded by SEQ ID NO:
1, 6, or 4; preferably the MybTF protein confers enhanced fungal
resistance relative to control plants; [0132] (iii) a nucleic acid
molecule which hybridizes with a complementary sequence of any of
the nucleic acid molecules of (i) or (ii) under high stringency
hybridization conditions; preferably encoding a MybTF protein;
preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0133] (iv) a nucleic acid encoding the same MybTF
protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0134] Preferred splice variants of SEQ ID NO: 1 or 8 consist of or
comprise anyone of the nucleotide sequences shown in SEQ ID NO: 4
or 6. Most preferred is the MybTF nucleic acid splice variant as
shown in SEQ ID NO: 6.
[0135] Preferably the MybTF nucleic acid is an isolated nucleic
acid molecule comprising a nucleic acid selected from the group
consisting of: [0136] (i) a nucleic acid having in increasing order
of at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the nucleic
acid sequence represented by SEQ ID NO: 1 or 8, or a splice variant
thereof; [0137] (ii) a nucleic acid molecule which hybridizes with
a complementary sequence of any of the nucleic acid molecules of
(i) under high stringency hybridization conditions; preferably
encoding a MybTF protein; preferably wherein the nucleic acid
molecule codes for a polypeptide which has essentially identical
properties to the polypeptide described in SEQ ID NO: 7 or 5;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; and [0138] (iii) a nucleic acid
encoding the same MybTF protein as the MybTF nucleic acids of (i)
to (ii) above, but differing from the MybTF nucleic acids of (i) to
(ii) above due to the degeneracy of the genetic code; wherein the
splice variant thereof is selected from the group consisting of:
[0139] (i) a nucleic acid having in increasing order of preference
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the nucleic
acid sequence represented by SEQ ID NO: 4 or 6, or a functional
fragment, derivative, orthologue, or paralogue thereof; [0140] (ii)
a nucleic acid encoding a MybTF protein having in increasing order
of preference at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% sequence identity
to the amino acid sequence represented by SEQ ID NO: 7 or 5, or a
functional fragment, derivative, orthologue, or paralogue thereof;
preferably the MybTF protein has essentially the same biological
activity as a MybTF protein encoded by SEQ ID NO: 1, 6, or 4;
preferably the MybTF protein confers enhanced fungal resistance
relative to control plants; [0141] (iii) a nucleic acid molecule
which hybridizes with a complementary sequence of any of the
nucleic acid molecules of (i) or (ii) under high stringency
hybridization conditions; preferably encoding a MybTF protein;
preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0142] (iv) a nucleic acid encoding the same MybTF
protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0143] More preferably the MybTF nucleic acid is an isolated
nucleic acid molecule comprising a nucleic acid selected from the
group consisting of:
[0144] a nucleic acid having in increasing order of preference
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to the nucleic acid sequence
represented by SEQ ID NO: 1 or 8, or a splice variant thereof;
wherein the splice variant thereof is selected from the group
consisting of:
[0145] a nucleic acid having in increasing order of preference at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to the nucleic acid sequence
represented by SEQ ID NO: 4 or 6, preferably SEQ ID NO:4.
[0146] All the nucleic acid sequences mentioned herein
(single-stranded and double-stranded DNA and RNA sequences, for
example cDNA and mRNA) can be produced in a known way by chemical
synthesis from the nucleotide building blocks, e.g. by fragment
condensation of individual overlapping, complementary nucleic acid
building blocks of the double helix. Chemical synthesis of
oligonucleotides can, for example, be performed in a known way, by
the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press,
New York, pages 896-897). The accumulation of synthetic
oligonucleotides and filling of gaps by means of the Klenow
fragment of DNA polymerase and ligation reactions as well as
general cloning techniques are described in Sambrook et al. (1989),
see below.
[0147] The MybTF nucleic acids described herein are useful in the
constructs, methods, plants, harvestable parts and products of the
invention.
[0148] MybTF Proteins
[0149] The MybTF protein is preferably of the R2R3-MYB family,
preferably defined by SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, or 56, or a fragment, homolog,
derivative, orthologue or paralogue thereof. Preferably, the MybTF
protein of the present invention is encoded by a nucleic acid,
which has at least 70% sequence identity, at least 80%, at least
90%, at least 92%, at least 95%, at least 97%, at least 98%, at
least 99% sequence identity, or even 100% sequence identity with
SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, or 55, or a functional fragment
thereof. More preferably, the MybTF protein of the present
invention has at least 70% sequence identity, at least 80%, at
least 90%, at least 92%, at least 95%, at least 97%, at least 98%,
at least 99% sequence identity, or even 100% sequence identity with
SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, or 56, or is a functional fragment thereof, an
orthologue or a paralogue thereof. Most preferred is at least 90
identity, at least 92%, at least 95%, at least 97% identity, more
preferred is at least 98% or at least 99% identity with SEQ ID NO:
7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
or 56.
[0150] More preferably, the MybTF protein of the present invention
has at least 70% sequence identity, at least 80%, at least 90%, at
least 92%, at least 95%, at least 97%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO: 7
or is a functional fragment thereof, an orthologue or a paralogue
thereof.
[0151] More preferably, the MybTF protein of the present invention
has at least 70% sequence identity, at least 80%, at least 90%, at
least 92%, at least 95%, at least 97%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
5, or is a functional fragment thereof, an orthologue or a
paralogue thereof.
[0152] The MybTF protein is preferably defined by SEQ ID NO: 7, 5,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56,
or a fragment, homolog, derivative, orthologue or paralogue
thereof. Preferably, the MybTF protein of the present invention is
encoded by a nucleic acid, which has at least 70% sequence
identity, at least 80%, at least 90%, at least 92%, at least 95%,
at least 97%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, or a
functional fragment thereof. More preferably, the MybTF protein of
the present invention has at least 70% sequence identity, at least
80%, at least 90%, at least 92%, at least 95%, at least 97%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, or 56, or is a functional fragment thereof,
an orthologue or a paralogue thereof. Most preferred is at least
92%, at least 95%, at least 97% identity, more preferred is at
least 98% or at least 99% identity with SEQ ID NO: 7, 5, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.
[0153] Preferably, the MybTF protein is a protein consisting of or
comprising an amino acid sequence selected from the group
consisting of: [0154] (i) an amino acid sequence having in
increasing order of preference at least 70%, at least 71%, at least
72%, at least 73%, at least 74%, at least 75%, at least 76%, at
least 77%, at least 78%, at least 79%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the amino acid sequence represented by SEQ ID
NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, or 56, or a functional fragment, derivative, orthologue, or
paralogue thereof; preferably the MybTF protein has essentially the
same biological activity as a MybTF protein encoded by SEQ ID NO:
2, 3, 1, 6, or 4; preferably the MybTF protein confers enhanced
fungal resistance relative to control plants; or [0155] (ii) an
amino acid sequence encoded by a nucleic acid having in increasing
order of preference at least 70%, at least 71%, at least 72%, at
least 73%, at least 74%, at least 75%, at least 76%, at least 77%,
at least 78%, at least 79%, at least 80%, at least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to the nucleic acid sequence represented by SEQ ID NO: 2,
3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55, or a functional fragment, derivative,
orthologue, or paralogue thereof, or a splice variant thereof;
preferably the MybTF protein confers enhanced fungal resistance
relative to control plants.
[0156] Preferably, the MybTF protein is a protein comprising an
amino acid sequence selected from the group consisting of: [0157]
(i) an amino acid sequence having in increasing order of preference
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the amino
acid sequence represented by SEQ ID NO: 7, or a functional
fragment, derivative, orthologue, or paralogue thereof; preferably
the MybTF protein has essentially the same biological activity as a
MybTF protein encoded by SEQ ID NO: 2, 6, or 1, preferably the
MybTF protein confers enhanced fungal resistance relative to
control plants; or [0158] (ii) an amino acid sequence encoded by a
nucleic acid having in increasing order of preference at least 70%,
at least 71%, at least 72%, at least 73%, at least 74%, at least
75%, at least 76%, at least 77%, at least 78%, at least 79%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to the nucleic acid sequence
represented by SEQ ID NO: 2, 6, 9-16, or 1, or a functional
fragment, derivative, orthologue, or paralogue thereof, or a splice
variant thereof; preferably the MybTF protein confers enhanced
fungal resistance relative to control plants.
[0159] Preferably, the MybTF protein is a protein comprising an
amino acid sequence selected from the group consisting of: [0160]
(i) an amino acid sequence having in increasing order of preference
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the amino
acid sequence represented by SEQ ID NO: 5, or a functional
fragment, derivative, orthologue, or paralogue thereof; preferably
the MybTF protein has essentially the same biological activity as a
MybTF protein encoded by SEQ ID NO: 3, 4, or 1; preferably the
MybTF protein confers enhanced fungal resistance relative to
control plants; or [0161] (ii) an amino acid sequence encoded by a
nucleic acid having in increasing order of preference at least 70%,
at least 71%, at least 72%, at least 73%, at least 74%, at least
75%, at least 76%, at least 77%, at least 78%, at least 79%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or 100% sequence identity to the nucleic acid sequence
represented by SEQ ID NO: 3, 4, 17-24, or 1, or a functional
fragment, derivative, orthologue, or paralogue thereof, or a splice
variant thereof; preferably the MybTF protein confers enhanced
fungal resistance relative to control plants.
[0162] A preferred derivative of a MybTF protein is a MybTF protein
consisting of or comprising an amino acid sequence selected from
the group consisting of:
[0163] an amino acid sequence having in increasing order of
preference at least 70%, at least 71%, at least 72%, at least 73%,
at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% sequence identity to the
amino acid sequence represented by SEQ ID NO: 7, 5, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, wherein the
non-identical amino acid residues are conservative amino acid
substitutions, preferably as shown in Table 1, of the corresponding
amino acid residue of SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, or 56; preferably the MybTF protein
has essentially the same biological activity as SEQ ID NO: 7 or 5,
or as a MybTF protein encoded by SEQ ID NO: 2, 3, 1, 6, or 4,
preferably the MybTF protein confers enhanced fungal resistance
relative to control plants.
[0164] Preferably, the MybTF protein consists of or comprises an
amino acid sequence represented by SEQ ID NO: 7, 5, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56 with one or more
conservative amino acid substitutions, preferably as shown in Table
1, of the corresponding amino acid residues of SEQ ID NO: 7, 5, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.
Preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
1-10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,
100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 60-170,
170-180, 180-190, 190-200, 200-210, or 210-220 amino acid residues
of SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, or 56 are conservative amino acid substitutions,
preferably as shown in Table 1, of the corresponding amino acid
residue of SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, or 56.
[0165] More preferably, the MybTF protein consists of or comprises
an amino acid sequence having at least 70%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity with an amino acid sequence as represented by SEQ
ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, or 56, wherein at least 1, at least 2, at least 3, at least
4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55, at least 60, at least 65, at least 70, at least 75, at
least 80, at least 85, at least 90, at least 95, at least 100, at
least 105, at least 110, at least 115, or at least 120 of the
non-identical amino acid residues, or wherein 1-10, 10-20, 20-30,
40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,
120-130, 130-140, 140-150, 150-160, 60-170, 170-180, 180-190,
190-200, 200-210, or 210-220 or even all of the nonidentical amino
acid residues are conservative amino acid substitutions, preferably
as shown in Table 1, of the corresponding amino acid residue of SEQ
ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, or 56.
[0166] Percentages of identity of a polypeptide or protein are
indicated with reference to the entire amino acid sequence
specifically disclosed herein.
[0167] Preferably, the MybTF protein comprises at least about 100,
at least about 150, at least about 200, at least about 225, at
least about 250, at least about 275, or at least about 300 amino
acid residues, preferably continuous amino acid residues,
preferably counted from the N-terminus or the C-terminus of the
amino acid sequence, or up to the full length of the amino acid
sequence set out in SEQ ID NO: 5, 42, 44, 46, 48, 50, 52, 54, or
56.
[0168] Preferably, the MybTF polypeptide comprises about 100-150,
about 150-200, about 200-225, about 225-250, about 250-275, about
275-300, or about 300-305 amino acid residues, preferably
consecutive amino acid residues, preferably counted from the
N-terminus or C-terminus of the amino acid sequence, or up to the
full length of any of the amino acid sequences encoded by the
nucleic acid sequences set out in SEQ ID NO: 5, 42, 44, 46, 48, 50,
52, 54, or 56.
[0169] Preferably, the MybTF protein comprises at least about 100,
at least about 125, at least about 150, at least about 175, at
least about 200, or at least about 225 amino acid residues,
preferably continuous amino acid residues, preferably counted from
the N-terminus or the C-terminus of the amino acid sequence, or up
to the full length of the amino acid sequence set out in SEQ ID NO:
7, 26, 28, 30, 32, 34, 36, 38, or 40.
[0170] Preferably, the MybTF polypeptide comprises about 100-125,
about 125-150, about 150-175, about 175-200, about 200-225, or
about 225-233 amino acid residues, preferably consecutive amino
acid residues, preferably counted from the N-terminus or C-terminus
of the amino acid sequence, or up to the full length of any of the
amino acid sequences encoded by the nucleic acid sequences set out
in SEQ ID NO: 7, 26, 28, 30, 32, 34, 36, 38, or 40.
[0171] The MybTF proteins described herein are useful in the
constructs, methods, plants, harvestable parts and products of the
invention.
[0172] Methods for Increasing Fungal Resistance; Methods for
Modulating Gene Expression
[0173] One embodiment of the invention is a method for increasing
fungal resistance, preferably resistance to Phacopsoracea, for
example soy bean rust, in a plant, plant part, or plant cell by
increasing the expression of a MybTF protein or a functional
fragment, orthologue, paralogue or homologue thereof in comparison
to wild-type plants, wild-type plant parts or wild-type plant
cells.
[0174] The present invention also provides a method for increasing
resistance to fungal pathogens, in particular a heminecrotrophic
pathogen, in particular to rust pathogens (i.e., fungal pathogens
of the order Pucciniales), preferably fungal pathogens of the
family Phacopsoraceae, preferably against fungal pathogens of the
genus Phacopsora, most preferably against Phakopsora pachyrhizi and
Phakopsora meibomiae, also known as soy bean rust in plants or
plant cells, wherein in comparison to wild type plants, wild type
plant parts, or wild type plant cells a MybTF protein is
overexpressed.
[0175] The present invention further provides a method for
increasing resistance to fungal pathogens of the genus Phacopsora,
most preferably against Phakopsora pachyrhizi and Phakopsora
meibomiae, also known as soy bean rust in plants or plant cells by
overexpression of a MybTF protein.
[0176] In preferred embodiments, the protein amount and/or function
of the MybTF protein in the plant is increased by at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, or at least 95% or more
in comparison to a wild type plant that is not transformed with the
MybTF nucleic acid.
[0177] In one embodiment of the invention, the MybTF protein is
encoded by a nucleic acid comprising [0178] (i) an exogenous
nucleic acid having at least 70%, for example at least 75%, more
preferably at least 80%, for example at least 85%, even more
preferably at least 90%, for example at least 95% or at least 96%
or at least 97% or at least 98% most preferably 99% identity with
SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, or 55, a functional fragment thereof,
or an orthologue or a paralogue thereof, or a splice variant
thereof; or by [0179] (ii) an exogenous nucleic acid encoding a
protein comprising an amino acid sequence having at least 70%, for
example at least 75%, more preferably at least 80%, for example at
least 85%, even more preferably at least 90%, for example at least
95% or at least 96% or at least 97% or at least 98% most preferably
99% homology with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, or 56, a functional fragment thereof,
an orthologue or a paralogue thereof, preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; [0180] (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; or by [0181] (iv) an
exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code.
[0182] A method for increasing fungal resistance, preferably
resistance to Phacopsoracea, for example soy bean rust, in a plant,
plant part, or plant cell, by increasing the expression of a MybTF
protein or a functional fragment, orthologue, paralogue or
homologue thereof, or a splice variant thereof, wherein the MybTF
protein is encoded by a nucleic acid comprising [0183] (i) an
exogenous nucleic acid having at least 70% sequence identity, at
least 80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55, or a functional fragment thereof, an
orthologue or a paralogue thereof, or a splice variant thereof;
[0184] (ii) an exogenous nucleic acid encoding a protein comprising
an amino acid sequence having at least 70% sequence identity, at
least 80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
or 56, a functional fragment thereof, an orthologue or a paralogue
thereof; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; [0185] (iii) an exogenous
nucleic acid capable of hybridizing under stringent conditions with
a complementary sequence of any of the nucleic acids according to
(i) or (ii); preferably encoding a MybTF protein; preferably
wherein the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0186] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code; is a further embodiment of the invention.
[0187] A method for increasing fungal resistance, preferably
resistance to Phacopsoracea, for example soy bean rust, in a plant,
plant part, or plant cell, by increasing the expression of a MybTF
protein or a functional fragment, orthologue, paralogue or
homologue thereof, or a splice variant thereof, wherein the MybTF
protein is encoded by [0188] (i) an exogenous nucleic acid having
at least 70% sequence identity, at least 80%, at least 90%, at
least 95%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 1 or a functional fragment
thereof, an orthologue or a paralogue thereof, or a splice variant
thereof; [0189] (ii) an exogenous nucleic acid encoding a protein
having at least 70% sequence identity, at least 80%, at least 90%,
at least 95%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 7 or 5, a functional
fragment thereof, an orthologue or a paralogue thereof; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; [0190] (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; and/or by [0191] (iv) an
exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code is a further embodiment of the invention.
[0192] A method for increasing fungal resistance, preferably
resistance to Phacopsoracea, for example soy bean rust, in a plant,
plant part, or plant cell, by increasing the expression of a MybTF
protein or a functional fragment, orthologue, paralogue or
homologue thereof, or a splice variant thereof, wherein the MybTF
protein is encoded by [0193] (i) an exogenous nucleic acid having
at least 70% sequence identity, at least 80%, at least 90%, at
least 95%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 2 or 6 or a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; [0194] (ii) an exogenous nucleic acid encoding a
protein having at least 70% sequence identity, at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 7, a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0195] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0196] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code is a further embodiment of the invention.
[0197] A method for increasing fungal resistance, preferably
resistance to Phacopsoracea, for example soy bean rust, in a plant,
plant part, or plant cell, by increasing the expression of a MybTF
protein or a functional fragment, orthologue, paralogue or
homologue thereof, or a splice variant thereof, wherein the MybTF
protein is encoded by [0198] (i) an exogenous nucleic acid having
at least 70% sequence identity, at least 80%, at least 90%, at
least 95%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 3 or 4 or a functional
fragment thereof, an orthologue or a paralogue thereof, or a splice
variant thereof; [0199] (ii) an exogenous nucleic acid encoding a
protein having at least 70% sequence identity, at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 5, a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0200] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0201] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code is a further embodiment of the invention.
[0202] In a further method of the invention, the method comprises
the steps of [0203] (a) stably transforming a plant cell with a
recombinant expression cassette comprising [0204] (i) a nucleic
acid having at least 70% sequence identity, at least 80%, at least
90%, at least 95%, at least 98%, at least 99% sequence identity, or
even 100% sequence identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55,
or a functional fragment thereof, or an orthologue or a paralogue
thereof, or a splice variant thereof; [0205] (ii) a nucleic acid
coding for a protein comprising an amino acid sequence having at
least 70% sequence identity, at least 80%, at least 90%, at least
95%, at least 98%, at least 99% sequence identity, or even 100%
sequence identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, or 56, a functional fragment
thereof, an orthologue or a paralogue thereof; preferably the
encoded protein confers enhanced fungal resistance relative to
control plants; [0206] (iii) a nucleic acid capable of hybridizing
under stringent conditions with a complementary sequence of any of
the nucleic acids according to (i) or (ii); preferably encoding a
MybTF protein; preferably wherein the nucleic acid molecule codes
for a polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0207] (iv) a nucleic acid encoding the same MybTF
polypeptide as the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code, in functional linkage with a
promoter; [0208] (b) regenerating the plant from the plant cell;
and [0209] (c) expressing said nucleic acid, optionally wherein the
nucleic acid which codes for a MybTF protein is expressed in an
amount and for a period sufficient to generate or to increase
soybean rust resistance in said plant.
[0210] Preferably, the method comprises the steps of [0211] (a)
stably transforming a plant cell with a recombinant expression
cassette comprising [0212] (i) a nucleic acid having at least 70%
sequence identity, at least 80%, at least 90%, at least 95%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 1, or a functional fragment thereof, or an
orthologue or a paralogue thereof, or a splice variant thereof;
[0213] (ii) a nucleic acid coding for a protein having at least 70%
sequence identity, at least 80%, at least 90%, at least 95%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 7 or 5, a functional fragment thereof, an
orthologue or a paralogue thereof; preferably the encoded protein
confers enhanced fungal resistance relative to control plants;
[0214] (iii) a nucleic acid capable of hybridizing under stringent
conditions with a complementary sequence of any of the nucleic
acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0215] (iv) a nucleic acid encoding the same MybTF
polypeptide as the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code, in functional linkage with a
promoter; [0216] (b) regenerating the plant from the plant cell;
and [0217] (c) expressing said nucleic acid, optionally wherein the
nucleic acid which codes for a
[0218] MybTF protein is expressed in an amount and for a period
sufficient to generate or to increase soybean rust resistance in
said plant.
[0219] Preferably, the method comprises the steps of [0220] (a)
stably transforming a plant cell with a recombinant expression
cassette comprising [0221] (i) a nucleic acid having at least 70%
sequence identity, at least 80%, at least 90%, at least 95%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 2 or 6, or a functional fragment thereof,
or an orthologue or a paralogue thereof, or a splice variant
thereof; [0222] (ii) a nucleic acid coding for a protein having at
least 70% sequence identity, at least 80%, at least 90%, at least
95%, at least 98%, at least 99% sequence identity, or even 100%
sequence identity with SEQ ID NO: 7, a functional fragment thereof,
an orthologue or a paralogue thereof; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; [0223] (iii) a nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0224] (iv) a nucleic acid encoding the same MybTF
polypeptide as the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code, in functional linkage with a
promoter; [0225] (b) regenerating the plant from the plant cell;
and [0226] (c) expressing said nucleic acid, optionally wherein the
nucleic acid which codes for a MybTF protein is expressed in an
amount and for a period sufficient to generate or to increase
soybean rust resistance in said plant.
[0227] Preferably, the method comprises the steps of [0228] (a)
stably transforming a plant cell with a recombinant expression
cassette comprising [0229] (i) a nucleic acid having at least 70%
sequence identity, at least 80%, at least 90%, at least 95%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 3 or 4, or a functional fragment thereof,
or an orthologue or a paralogue thereof, or a splice variant
thereof; [0230] (ii) a nucleic acid coding for a protein having at
least 70% sequence identity, at least 80%, at least 90%, at least
95%, at least 98%, at least 99% sequence identity, or even 100%
sequence identity with SEQ ID NO: 5, a functional fragment thereof,
an orthologue or a paralogue thereof; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; [0231] (iii) a nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0232] (iv) a nucleic acid encoding the same MybTF
polypeptide as the nucleic acids of (i) to (iii) above, but
differing from the nucleic acids of (i) to (iii) above due to the
degeneracy of the genetic code, in functional linkage with a
promoter; [0233] (b) regenerating the plant from the plant cell;
and [0234] (c) expressing said nucleic acid, optionally wherein the
nucleic acid which codes for a MybTF protein is expressed in an
amount and for a period sufficient to generate or to increase
soybean rust resistance in said plant.
[0235] Preferably, the promoter is a rust induced and/or
mesophyll-specific promoter, preferably the rust induced mesophyll
specific promoter 820.
[0236] Preferably, the method for increasing fungal resistance,
preferably resistance to Phacopsoracea, for example soy bean rust,
in a plant, plant part, or plant cell further comprises the step of
selecting a transgenic plant expressing [0237] (i) an exogenous
nucleic acid having at least 70% sequence identity, at least 80%,
at least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 2, 3, 1,
6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, or 55, or a functional fragment thereof, or an orthologue
or a paralogue thereof, or a splice variant thereof; [0238] (ii) an
exogenous nucleic acid coding for a protein having at least 70%
sequence identity, at least 80%, at least 90%, at least 95%, at
least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, or 56, a functional fragment thereof, an
orthologue or a paralogue thereof; preferably the encoded protein
confers enhanced fungal resistance relative to control plants;
[0239] (iii) an exogenous nucleic acid capable of hybridizing under
stringent conditions a complementary sequence of any of the nucleic
acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0240] (iv) an exogenous nucleic acid encoding the
same MybTF polypeptide as the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0241] A preferred embodiment is a method for increasing resistance
to soy bean rust in a soy bean plant, soy bean plant part, or soy
bean plant cell, by increasing the expression of a MybTF protein,
wherein the MybTF protein is encoded by a nucleic acid comprising
[0242] (i) an exogenous nucleic acid having at least 80%, at least
90%, at least 95%, at least 98%, at least 99% sequence identity, or
even 100% sequence identity with SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55;
[0243] (ii) an exogenous nucleic acid encoding a protein comprising
an amino acid sequence having at least 80%, at least 90%, at least
95%, at least 98%, at least 99% sequence identity, or even 100%
sequence identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, or 56; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; [0244] (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; and/or by [0245] (iv) an
exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code, wherein increasing the expression of the MybTF protein is
achieved by transforming the soy bean plant, plant part or plant
cell with a nucleic acid comprising the nucleic acid set out under
item (i) or (ii) or (iii) or (iv).
[0246] Also a preferred embodiment is a method for increasing
resistance to soy bean rust in a soy bean plant, soy bean plant
part, or soy bean plant cell, by increasing the expression of a
MybTF protein, wherein the MybTF protein is encoded by a nucleic
acid comprising [0247] (i) an exogenous nucleic acid having at
least 80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55; [0248] (ii) an exogenous nucleic acid
encoding a protein comprising an amino acid sequence having at
least 80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
or 56; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; or [0249] (iii) an exogenous
nucleic acid encoding the same MybTF protein as the nucleic acids
of (i) to (ii) above, but differing from the nucleic acids of (i)
to (ii) above due to the degeneracy of the genetic code, wherein
increasing the expression of the MybTF protein is achieved by
transforming the soy bean plant, plant part or plant cell with a
nucleic acid comprising the nucleic acid set out under item (i) or
(ii) or (iii).
[0250] The fungal pathogens or fungus-like pathogens (such as, for
example, Chromista) can belong to the group comprising
Plasmodiophoramycota, Oomycota, Ascomycota, Chytridiomycetes,
Zygomycetes, Basidiomycota or Deuteromycetes (Fungi imperfecti).
Pathogens which may be mentioned by way of example, but not by
limitation, are those detailed in Tables 2 and 3, and the diseases
which are associated with them.
TABLE-US-00005 TABLE 2 Diseases caused by biotrophic and/or
heminecrotrophic phytopathogenic fungi Disease Pathogen Leaf rust
Puccinia recondita Yellow rust P. striiformis Powdery mildew
Erysiphe graminis/Blumeria graminis Rust (common corn) Puccinia
sorghi Rust (Southern corn) Puccinia polysora Tobacco leaf spot
Cercospora nicotianae Rust (soybean) Phakopsora pachyrhizi, P.
meibomiae Rust (tropical corn) Physopella pallescens, P. zeae =
Angiopsora zeae
TABLE-US-00006 TABLE 3 Diseases caused by necrotrophic and/or
hemibiotrophic fungi and Oomycetes Disease Pathogen Plume blotch
Septoria (Stagonospora) nodorum Leaf blotch Septoria tritici Ear
fusarioses Fusarium spp. Late blight Phytophthora infestans
Anthrocnose Colletotrichum graminicola (teleomorph: Glomerella leaf
blight graminicola Politis); Glomerella tucumanensis Anthracnose
(anamorph: Glomerella falcatum Went) stalk rot Curvularia
Curvularia clavata, C. eragrostidis, = C. maculans leaf spot
(teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C.
intermedia (teleomorph: Cochliobolus intermedius), Curvularia
lunata (teleomorph: Cochliobolus lunatus), Curvularia pallescens
(teleomorph: Cochliobolus pallescens), Curvularia senegalensis, C.
tuberculata (teleomorph: Cochliobolus tuberculatus) Didymella
Didymella exitalis leaf spot Diplodia leaf Stenocarpella macrospora
= spot or streak Diplodialeaf macrospora Brown stripe Sclerophthora
rayssiae var. zeae downy mildew Crazy top Sclerophthora macrospora
= downy mildew Sclerospora macrospora Green ear Sclerospora
graminicola downy mildew (graminicola downy mildew) Leaf spots,
Alternaria alternata, minor Ascochyta maydis, A. tritici, A.
zeicola, Bipolaris victoriae = Helminthosporium victoriae
(teleomorph: Cochliobolus victoriae), C. sativus (anamorph:
Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum
nigrum, Exserohilum prolatum = Drechslera prolata (teleomorph:
Setosphaeria prolata) Graphium penicillioides, Leptosphaeria
maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph:
Scolecosporiella sp.), Paraphaeosphaeria michotii, Phoma sp.,
Septoria zeae, S. zeicola, S. zeina Northern corn Setosphaeria
turcica (anamorph: Exserohilum turcicum = leaf blight
Helminthosporium turcicum) (white blast, crown stalk rot, stripe)
Northern corn Cochliobolus carbonum (anamorph: Bipolaris zeicola =
leaf spot Helminthosporium carbonum) Helmintho- sporium ear rot
(race 1) Phaeosphaeria Phaeosphaeria maydis = Sphaerulina maydis
leaf spot Rostratum Setosphaeria rostrata, (anamorph: leaf spot
xserohilum rostratum = Helminthosporium rostratum) (Helmintho-
sporium leaf disease, ear and stalk rot) Java downy
Peronosclerospora maydis = mildew Sclerospora maydis Philippine
Peronosclerospora philippinensis = downy mildew Sclerospora
philippinensis Sorghum Peronosclerospora sorghi = downy mildew
Sclerospora sorghi Spontaneum Peronosclerospora spontanea = downy
mildew Sclerospora spontanea Sugarcane Peronosclerospora sacchari =
downy mildew Sclerospora sacchari Sclerotium Sclerotium rolfsii
Sacc. (teleomorph: Athelia rolfsii) ear rot (southern blight) Seed
rot- Bipolaris sorokiniana, B. zeicola = Helminthosporium seedling
carbonum, Diplodia maydis, Exserohilum pedicillatum, blight
Exserohilum turcicum = Helminthosporium turcicum, Fusarium
avenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph:
F. graminearum), Macrophomina phaseolina, Penicillium spp.,
Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae,
Sclerotium rolfsii, Spicaria sp. Selenophoma Selenophoma sp. leaf
spot Yellow leaf Ascochyta ischaemi, Phyllosticta maydis
(teleomorph: blight Mycosphaerella zeae-maydis) Zonate leaf
Gloeocercospora sorghi spot
[0251] The following are especially preferred: [0252]
Plasmodiophoromycota such as Plasmodiophora brassicae (clubroot of
crucifers), Spongospora subterranea, Polymyxa graminis, [0253]
Oomycota such as Bremia lactucae (downy mildew of lettuce),
Peronospora (downy mildew) in snapdragon (P. antirrhini), onion (P.
destructor), spinach (P. effusa), soybean (P. manchurica), tobacco
("blue mold"; P. tabacina) alfalfa and clover (P. trifolium),
Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy
mildew in grapevines) (P. viticola) and sunflower (P. halstedii),
Sclerophthora macrospora (downy mildew in cereals and grasses),
Pythium (for example damping-off of Beta beet caused by P.
debaryanum), Phytophthora infestans (late blight in potato and in
tomato and the like), Albugo spec. [0254] Ascomycota such as
Microdochium nivale (snow mold of rye and wheat), Fusarium,
Fusarium graminearum, Fusarium culmorum (partial ear sterility
mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato),
Blumeria graminis (powdery mildew of barley (f.sp. hordei) and
wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea),
Nectria galligena (Nectria canker of fruit trees), Uncinula necator
(powdery mildew of grapevine), Pseudopeziza tracheiphila (red fire
disease of grapevine), Claviceps purpurea (ergot on, for example,
rye and grasses), Gaeumannomyces graminis (take-all on wheat, rye
and other grasses), Magnaporthe grisea, Pyrenophora graminea (leaf
stripe of barley), Pyrenophora teres (net blotch of barley),
Pyrenophora tritici-repentis (leaf blight of wheat), Venturia
inaequalis (apple scab), Sclerotinia sclerotium (stalk break, stem
rot), Pseudopeziza medicaginis (leaf spot of alfalfa, white and red
clover). [0255] Basidiomycetes such as Typhula incarnata (typhula
blight on barley, rye, wheat), Ustilago maydis (blister smut on
maize), Ustilago nuda (loose smut on barley), Ustilago tritici
(loose smut on wheat, spelt), Ustilago avenae (loose smut on oats),
Rhizoctonia solani (rhizoctonia root rot of potato), Sphacelotheca
spp. (head smut of sorghum), Melampsora lini (rust of flax),
Puccinia graminis (stem rust of wheat, barley, rye, oats), Puccinia
recondita (leaf rust on wheat), Puccinia dispersa (brown rust on
rye), Puccinia hordei (leaf rust of barley), Puccinia coronata
(crown rust of oats), Puccinia striiformis (yellow rust of wheat,
barley, rye and a large number of grasses), Uromyces appendiculatus
(brown rust of bean), Sclerotium rolfsii (root and stem rots of
many plants). [0256] Deuteromycetes (Fungi imperfecti) such as
Septoria (Stagonospora) nodorum (glume blotch) of wheat (Septoria
tritici), Pseudocercosporella herpotrichoides (eyespot of wheat,
barley, rye), Rynchosporium secalis (leaf spot on rye and barley),
Alternaria solani (early blight of potato, tomato), Phoma betae
(blackleg on Beta beet), Cercospora beticola (leaf spot on Beta
beet), Alternaria brassicae (black spot on oilseed rape, cabbage
and other crucifers), Verticillium dahliae (verticillium wilt),
Colletotrichum, Colletotrichum lindemuthianum (bean anthracnose),
Phoma lingam (blackleg of cabbage and oilseed rape), Botrytis
cinerea (grey mold of grapevine, strawberry, tomato, hops and the
like).
[0257] Especially preferred are biotrophic pathogens, more
preferably heminecrotrophic pathogens, e.g., Phakopsora pachyrhizi
and/or those pathogens which have essentially a similar infection
mechanism as Phakopsora pachyrhizi, as described herein.
Particularly preferred are pathogens from the subclass
Pucciniomycetes, preferably from the order Pucciniales (rust),
previously known as Uredinales, among which in particular the
Melompsoraceae. Preferred are Phakopsoraceae, more preferably
Phakopsora. Especially preferred are Phakopsora pachyrhizi and/or
Phakopsora meibomiae.
[0258] Also preferred rust fungi are selected from the group of
Puccinia, Gymnosporangium, Juniperus, Cronartium, Hemileia, and
Uromyces; preferably Puccinia sorghi, Gymnosporangium
juniperi-virginianae, Juniperus virginiana, Cronartium ribicola,
Hemlleia vastatrix, Puccinia graminis, Puccinia coronata, Uromyces
phaseoli, Puccinia hemerocallidis, Puccinia persistens subsp.
Triticina, Puccinia striiformis, Puccinia graminis causes, and/or
Uromyces appendeculatus.
[0259] Further preferred pathogens, preferably pathogens of maize,
are pathogens causing stalk rot diseases, in particular Fusarium
stalk rot, Gibberella stalk rot, Diplodia stalk rot, and Charcoal
rot and pathogens causing anthracnose. Preferred pathogens causing
Fusarium stalk rot are Fusarium verticillioides, Fusarium
proliferatum or Fusarium subglutinans. A preferred pathogen causing
Gibberella stalk rot is Fusarium graminearum. A preferred pathogen
causing Diplodia stalk rot is Diplodia maydis. A preferred pathogen
causing Charcoal rot is Macrophomina phaseollna. A preferred
pathogen causing anthracnose is Colletotrichum graminicola.
[0260] MybTF Expression Constructs and Vector Constructs
[0261] A recombinant vector construct comprising: [0262] (a) (i) a
nucleic acid having at least 70% sequence identity, at least 80%,
at least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 2, 3, 1,
6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, or 55, or a functional fragment thereof, or an orthologue
or a paralogue thereof, or a splice variant thereof; [0263] (ii) a
nucleic acid coding for a protein comprising an amino acid sequence
having at least 70% sequence identity, at least 80%, at least 90%,
at least 95%, at least 98%, at least 99% sequence identity, or even
100% sequence identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, a functional
fragment thereof, an orthologue or a paralogue thereof; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; [0264] (iii) a nucleic acid capable of hybridizing
under stringent conditions with a complementary sequence of any of
the nucleic acids according to (i) or (ii); preferably encoding a
MybTF protein; preferably wherein the nucleic acid molecule codes
for a polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0265] (iv) a nucleic acid encoding the same MybTF
protein as the nucleic acids of (i) to (iii) above, but differing
from the nucleic acids of (i) to (iii) above due to the degeneracy
of the genetic code, operably linked with [0266] (b) a promoter and
[0267] (c) a transcription termination sequence is a further
embodiment of the invention.
[0268] Furthermore, a recombinant vector construct is provided
comprising: [0269] (a) (i) a nucleic acid having at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 2, 3, 1,
6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, or 55; [0270] (ii) a nucleic acid coding for a protein
comprising an amino acid sequence having at least 80%, at least
90%, at least 95%, at least 98%, at least 99% sequence identity, or
even 100% sequence identity with SEQ ID NO: 7, 5, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56; preferably the
encoded protein confers enhanced fungal resistance relative to
control plants; [0271] (iii) a nucleic acid capable of hybridizing
under stringent conditions with a complementary sequence of any of
the nucleic acids according to (i) or (ii); preferably encoding a
MybTF protein; preferably wherein the nucleic acid molecule codes
for a polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or [0272] (iv) a nucleic acid encoding the same MybTF
protein as the nucleic acids of (i) to (iii) above, but differing
from the nucleic acids of (i) to (iii) above due to the degeneracy
of the genetic code, operably linked with [0273] (b) a promoter and
[0274] (c) a transcription termination sequence is a further
embodiment of the invention.
[0275] Furthermore, a recombinant vector construct is provided
comprising: [0276] (a) (i) a nucleic acid having at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 1, [0277]
(ii) a nucleic acid coding for a protein having at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 7 or 5;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0278] (iii) a nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; and/or [0279] (iv) a nucleic
acid encoding the same MybTF protein as the nucleic acids of (i) to
(iii) above, but differing from the nucleic acids of (i) to (iii)
above due to the degeneracy of the genetic code, operably linked
with [0280] (b) a promoter and [0281] (c) a transcription
termination sequence is a further embodiment of the invention.
[0282] Furthermore, a recombinant vector construct is provided
comprising: [0283] (a) (i) a nucleic acid having at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 2 or 6;
[0284] (ii) a nucleic acid coding for a protein having at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
7; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; [0285] (iii) a nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or [0286] (iv) a
nucleic acid encoding the same MybTF protein as the nucleic acids
of (i) to (iii) above, but differing from the nucleic acids of (i)
to (iii) above due to the degeneracy of the genetic code, operably
linked with [0287] (b) a promoter and [0288] (c) a transcription
termination sequence is a further embodiment of the invention.
[0289] Furthermore, a recombinant vector construct is provided
comprising: [0290] (a) (i) a nucleic acid having at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 3 or 4;
[0291] (ii) a nucleic acid coding for a protein having at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; [0292] (iii) a nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or [0293] (iv) a
nucleic acid encoding the same MybTF protein as the nucleic acids
of (i) to (iii) above, but differing from the nucleic acids of (i)
to (iii) above due to the degeneracy of the genetic code, operably
linked with [0294] (b) a promoter and [0295] (c) a transcription
termination sequence is a further embodiment of the invention.
[0296] In the case of a genomic library, the natural genetic
environment of the nucleic acid sequence is preferably retained, at
least in part. The environment preferably flanks the nucleic acid
sequence at least on one side and has a sequence length of at least
50 bp, preferably at least 500 bp, especially preferably at least
1000 bp, most preferably at least 5000 bp.
[0297] Promoters according to the present invention may be
constitutive, inducible, in particular pathogen-inducible,
developmental stage-preferred, cell type-preferred,
tissue-preferred or organ-preferred. Constitutive promoters are
active under most conditions. Non-limiting examples of constitutive
promoters include the CaMV 19S and 35S promoters (Odell et al.,
1985, Nature 313:810-812), the sX CaMV 35S promoter (Kay et al.,
1987, Science 236:1299-1302), the Sep1 promoter, the rice actin
promoter (McElroy et al., 1990, Plant Cell 2:163-171), the
Arabidopsis actin promoter, the ubiquitin promoter (Christensen et
al., 1989, Plant Molec. Biol. 18:675-689); pEmu (Last et al., 1991,
Theor. Appl. Genet. 81:581-588), the figwort mosaic virus 35S
promoter, the Smas promoter (Velten et al., 1984, EMBO J.
3:2723-2730), the GRP1-8 promoter, the cinnamyl alcohol
dehydrogenase promoter (U.S. Pat. No. 5,683,439), promoters from
the T-DNA of Agrobacterium, such as mannopine synthase, nopaline
synthase, and octopine synthase, the small subunit of ribulose
biphosphate carboxylase (ssuRUBISCO) promoter, and/or the like.
[0298] Preferably, the expression vector of the invention comprises
a constitutive promoter, mesophyll-specific promoter,
epidermis-specific promoter, root-specific promoter, a pathogen
inducible promoter, or a fungal-inducible promoter.
[0299] A promoter is inducible, if its activity, measured on the
amount of RNA produced under control of the promoter, is at least
30%, at least 40%, at least 50% preferably at least 60%, at least
70%, at least 80%, at least 90% more preferred at least 100%, at
least 200%, at least 300% higher in its induced state, than in its
un-induced state. A promoter is cell-, tissue- or organ-specific,
if its activity, measured on the amount of RNA produced under
control of the promoter, is at least 30%, at least 40%, at least
50% preferably at least 60%, at least 70%, at least 80%, at least
90% more preferred at least 100%, at least 200%, at least 300%
higher in a particular cell-type, tissue or organ, then in other
cell-types or tissues of the same plant, preferably the other
cell-types or tissues are cell types or tissues of the same plant
organ, e.g. a root. In the case of organ specific promoters, the
promoter activity has to be compared to the promoter activity in
other plant organs, e.g. leaves, stems, flowers or seeds.
Preferably, the promoter is a constitutive promoter,
mesophyll-specific promoter, or epidermis-specific promoter.
[0300] In preferred embodiments, the increase in the protein amount
and/or activity of the MybTF protein takes place in a constitutive
or tissue-specific manner. In especially preferred embodiments, an
essentially pathogen-induced increase in the protein amount and/or
protein activity takes place, for example by recombinant expression
of the MybTF nucleic acid under the control of a fungal-inducable
promoter. In particular, the expression of the MybTF nucleic acid
takes place on fungal infected sites, where, however, preferably
the expression of the MybTF nucleic acid remains essentially
unchanged in tissues not infected by fungus.
[0301] Developmental stage-preferred promoters are preferentially
expressed at certain stages of development. Tissue and organ
preferred promoters include those that are preferentially expressed
in certain tissues or organs, such as leaves, roots, seeds, or
xylem. Examples of tissue preferred and organ preferred promoters
include, but are not limited to fruitpreferred, ovule-preferred,
male tissue-preferred, seed-preferred, integument-preferred,
tuber-preferred, stalk-preferred, pericarp-preferred,
leaf-preferred, stigma-preferred, pollenpreferred,
anther-preferred, a petal-preferred, sepal-preferred,
pedicel-preferred, siliquepreferred, stem-preferred, root-preferred
promoters and/or the like. Seed preferred promoters are
preferentially expressed during seed development and/or
germination. For example, seed preferred promoters can be
embryo-preferred, endosperm preferred and seed coatpreferred. See
Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred
promoters include, but are not limited to cellulose synthase
(celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1)
and/or the like.
[0302] Other suitable tissue-preferred or organ-preferred promoters
include, but are not limited to, the napin-gene promoter from
rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia
faba (Baeumlein et al., 1991, Mol Gen Genet. 225(3):459-67), the
oleosinpromoter from Arabidopsis (PCT Application No. WO 98/45461),
the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No.
5,504,200), the Bce4-promoter from Brassica (PCT Application No. WO
91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al.,
1992, Plant Journal, 2(2):233-9), as well as promoters conferring
seed specific expression in monocot plants like maize, barley,
wheat, rye, rice, etc. Suitable promoters to note are the Ipt2 or
Ipt1-gene promoter from barley (PCT Application No. WO 95/15389 and
PCT Application No. WO 95/23230) or those described in PCT
Application No. WO 99/16890 (promoters from the barley
hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin
gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene,
Sorghum kasirin-gene, and/or rye secalin gene).
[0303] Promoters useful according to the invention include, but are
not limited to, are the major chlorophyll a/b binding protein
promoter, histone promoters, the Ap3 promoter, the 3-conglycin
promoter, the napin promoter, the soybean lectin promoter, the
maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein
promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2,
bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the
maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085
and 5,545,546), the SGB6 promoter (U.S. Pat. No. 5,470,359), as
well as synthetic or other natural promoters.
[0304] Epidermis-spezific promoters may be selected from the group
consisting of: WIR5 (=GstA1); acc. X56012; Dudler &
Schweizer,
[0305] GLP4, acc. AJ310534; Wei Y., Zhang Z., Andersen C. H.,
Schmelzer E., Gregersen P. L., Collinge D. B., Smedegaard-Petersen
V. and Thordal-Christensen H., Plant Molecular Biology 36, 101
(1998),
[0306] GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and
Dudler R., Plant J. 20, 541 (1999); Prx7, acc. AJ003141, Kristensen
B. K., Ammitzboll H., Rasmussen S. K. and Nielsen K. A., Molecular
Plant Pathology, 2(6), 311 (2001);
[0307] GerA, acc. AF250933; Wu S., Druka A., Horvath H., Kleinhofs
A., Kannangara G. and von
[0308] Wettstein D., Plant Phys Biochem 38, 685 (2000);
[0309] OsROC1, acc. AP004656
[0310] RTBV, acc. AAV62708, AAV62707; Kloti A., Henrich C., Bieri
S., He X., Chen G., Burkhardt P. K., Wunn J., Lucca P., Hohn T.,
Potrykus I. and Futterer J., PMB 40, 249 (1999); Chitinase
ChtC2-Promoter from potato (Ancillo et al., Planta. 217(4), 566,
(2003));
[0311] AtProT3 Promoter (Grallath et al., Plant Physiology. 137(1),
117 (2005)); SHN-Promoters from Arabidopsis (AP2/EREBP
transcription factors involved in cutin and wax production) (Aaron
et al., Plant Cell. 16(9), 2463 (2004)); and/or GSTA1 from wheat
(Dudler et al., WP2005306368 and Altpeter et al., Plant Molecular
Biology. 57(2), 271 (2005)).
[0312] Mesophyll-specific promoters may be selected from the group
consisting of:
[0313] PPCZm1 (=PEPC); Kausch A. P., Owen T. P., Zachwieja S. J.,
Flynn A. R. and Sheen J., Plant Mol. Biol. 45, 1 (2001);
[0314] OsrbcS, Kyozuka et al., PlaNT Phys 102, 991 (1993); Kyozuka
J., McElroy D., Hayakawa T., Xie Y., Wu R. and Shimamoto K., Plant
Phys. 102, 991 (1993); OsPPDK, acc. AC099041;
[0315] TaGF-2.8, acc. M63223; Schweizer P., Christoffel A. and
Dudler R., Plant J. 20, 541 (1999);
[0316] TaFBPase, acc. X53957;
[0317] TaWIS1, acc. AF467542; US 200220115849;
[0318] HvBIS1, acc. AF467539; US 200220115849;
[0319] ZmMIS1, acc. AF467514; US 200220115849;
[0320] HvPR1a, acc. X74939; Bryngelsson et al., Mol. Plant Microbe
Interacti. 7 (2), 267 (1994);
[0321] HvPR1b, acc. X74940; Bryngelsson et al., Mol. Plant Microbe
Interact. 7(2), 267 (1994);
[0322] HvB1,3gluc; acc. AF479647;
[0323] HvPrx8, acc. AJ276227; Kristensen et al., Molecular Plant
Pathology, 2(6), 311 (2001); and/or
[0324] HvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C. H.,
Schmelzer E., Gregersen P. L., Collinge D. B., Smedegaard-Petersen
V. and Thordal-Christensen H. Plant Molecular Biology 36, 101
(1998).
[0325] Constitutive promoters may be selected from the group
consisting of [0326] PcUbi promoter from parsley (WO 03/102198)
[0327] CaMV 35S promoter: Cauliflower Mosaic Virus 35S promoter
(Benfey et al. 1989 EMBO J. 8(8): 2195-2202), [0328] STPT promoter:
Arabidopsis thaliana Short Triose phosphate translocator promoter
(Accession NM.sub.--123979) [0329] Act1 promoter: Oryza sativa
actin 1 gene promoter (McElroy et al. 1990 PLANT CELL 2(2) 163-171
a) and/or [0330] EF1A2 promoter: Glycine max translation elongation
factor EF1 alpha (US 20090133159).
[0331] In preferred embodiments, the increase in the protein
quantity or function of the MybTF protein takes place in a
constitutive or tissue-specific manner. In especially preferred
embodiments, an essentially pathogen-induced increase in the
protein quantity or protein function takes place, for example by
exogenous expression of the MybTF nucleic acid under the control of
a fungal-inducible promoter, preferably a rust-inducible promoter.
In particular, the expression of the MybTF nucleic acid takes place
on fungal infected sites, where, however, preferably the expression
of the MybTF nucleic acid sequence remains essentially unchanged in
tissues not infected by fungus.
[0332] Preferably, the MybTF nucleic acid is under the control of a
rust induced mesophyll specific promoter. More preferably, the
promoter is the rust induced mesophyll specific promoter 820.
[0333] A preferred terminator is the terminator of the cathepsin D
inhibitor gene from Solanum tuberosum.
[0334] Preferred promoter-terminator combinations with the gene of
interest inbetween are a promoter from parsley, preferably, the
parsley ubiquitine promoter, in combination with the terminator of
the cathepsin D inhibitor gene from Solanum tuberosum. Another
preferred promoter-terminator combination is the rust induced
mesophyll specific promoter 820 in combination with the terminator
of the cathepsin D inhibitor gene from Solanum tuberosum.
[0335] An intron sequence may also be added to the 5' untranslated
region (UTR) and/or the coding sequence of the partial coding
sequence to increase the amount of the mature message that
accumulates in the cytosol. Inclusion of a spliceable intron in the
transcription unit in both plant and animal expression constructs
has been shown to increase gene expression at both the mRNA and
protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell
biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200).
Such intron enhancement of gene expression is typically greatest
when placed near the 5' end of the transcription unit. Use of the
maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are
known in the art. For general information see: The Maize Handbook,
Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
[0336] One type of vector construct is a "plasmid," which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vecfor constructs are capable of autonomous
replication in a host plant cell into which they are introduced.
Other vector constructs are integrated into the genome of a host
plant cell upon introduction into the host cell, and thereby are
replicated along with the host genome. In particular the vector
construct is capable of directing the expression of gene to which
the vectors is operatively linked. However, the invention is
intended to include such other forms of expression vector
constructs, such as viral vectors (e.g., potato virus X, tobacco
rattle virus, and/or Gemini virus), which serve equivalent
functions.
[0337] Transgenic Organisms; Transgenic Plants, Plant Parts, and
Plant Cells
[0338] A preferred embodiment is a transgenic plant, transgenic
plant part, or transgenic plant cell overexpressing an exogenous
MybTF protein, preferably the transgenic plant comprises a
recombinant expression construct encoding the MybTF protein.
Preferably, the MybTF protein overexpressed in the plant, plant
part or plant cell is encoded by a nucleic acid comprising [0339]
(i) an exogenous nucleic acid having at least 70% identity with SEQ
ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, or 55, or a functional fragment, thereof,
an orthologue or a paralogue thereof, or a splice variant thereof;
or by [0340] (ii) an exogenous nucleic acid encoding a protein
comprising an amino acid sequence having at least 70% identity with
SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, or 56, a functional fragment thereof, an orthologue or
a paralogue thereof; preferably the encoded protein confers
enhanced fungal resistance relative to control plants; [0341] (iii)
an exogenous nucleic acid capable of hybridizing under stringent
conditions with a complementary sequence of any of the nucleic
acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or by [0342] (iv) an exogenous nucleic acid encoding
the same MybTF protein as the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0343] Most preferably, the exogenous nucleic acid has at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55; or comprises an exogenous nucleic acid
encoding a protein having at least 80%, at least 90%, at least 95%,
at least 98%, at least 99% sequence identity, or even 100% sequence
identity with SEQ ID NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, or 56.
[0344] A preferred embodiment is a transgenic plant, transgenic
plant part, or transgenic plant cell overexpressing an exogenous
MybTF protein. Preferably, the MybTF protein overexpressed in the
plant, plant part or plant cell is encoded by [0345] (i) an
exogenous nucleic acid having at least 70% identity with SEQ ID NO:
1 or a functional fragment, thereof, an orthologue or a paralogue
thereof, or a splice variant thereof; or by [0346] (ii) an
exogenous nucleic acid encoding a protein having at least 70%
identity with SEQ ID NO: 7 or 5, a functional fragment thereof, an
orthologue or a paralogue thereof; preferably the encoded protein
confers enhanced fungal resistance relative to control plants;
[0347] (iii) an exogenous nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or by [0348] (iv) an exogenous nucleic acid encoding
the same MybTF protein as the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0349] A preferred embodiment is a transgenic plant, transgenic
plant part, or transgenic plant cell overexpressing an exogenous
MybTF protein. Preferably, the MybTF protein overexpressed in the
plant, plant part or plant cell is encoded by [0350] (i) an
exogenous nucleic acid having at least 70% identity with SEQ ID NO:
2 or 6 or a functional fragment, thereof, an orthologue or a
paralogue thereof, or a splice variant thereof; or by [0351] (ii)
an exogenous nucleic acid encoding a protein having at least 70%
identity with SEQ ID NO: 7, a functional fragment thereof, an
orthologue or a paralogue thereof; preferably the encoded protein
confers enhanced fungal resistance relative to control plants;
[0352] (iii) an exogenous nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or by [0353] (iv) an exogenous nucleic acid encoding
the same MybTF protein as the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0354] A preferred embodiment is a transgenic plant, transgenic
plant part, or transgenic plant cell overexpressing an exogenous
MybTF protein. Preferably, the MybTF protein overexpressed in the
plant, plant part or plant cell is encoded by [0355] (i) an
exogenous nucleic acid having at least 70% identity with SEQ ID NO:
3 or 4 or a functional fragment, thereof, an orthologue or a
paralogue thereof, or a splice variant thereof; or by [0356] (ii)
an exogenous nucleic acid encoding a protein having at least 70%
identity with SEQ ID NO: 5, a functional fragment thereof, an
orthologue or a paralogue thereof; preferably the encoded protein
confers enhanced fungal resistance relative to control plants;
[0357] (iii) an exogenous nucleic acid capable of hybridizing under
stringent conditions with a complementary sequence of any of the
nucleic acids according to (i) or (ii); preferably encoding a MybTF
protein; preferably wherein the nucleic acid molecule codes for a
polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and/or by [0358] (iv) an exogenous nucleic acid encoding
the same MybTF protein as the nucleic acids of (i) to (iii) above,
but differing from the nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code.
[0359] Most preferably, the exogenous nucleic acid has at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO:
1; or comprises an exogenous nucleic acid encoding a protein having
at least 80%, at least 90%, at least 95%, at least 98%, at least
99% sequence identity, or even 100% sequence identity with SEQ ID
NO: 7 or 5.
[0360] Most preferably, the exogenous nucleic acid has at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO: 2
or 6; or comprises an exogenous nucleic acid encoding a protein
having at least 80%, at least 90%, at least 95%, at least 98%, at
least 99% sequence identity, or even 100% sequence identity with
SEQ ID NO: 7.
[0361] Most preferably, the exogenous nucleic acid has at least
80%, at least 90%, at least 95%, at least 98%, at least 99%
sequence identity, or even 100% sequence identity with SEQ ID NO: 3
or 4; or comprises an exogenous nucleic acid encoding a protein
having at least 80%, at least 90%, at least 95%, at least 98%, at
least 99% sequence identity, or even 100% sequence identity with
SEQ ID NO: 5.
[0362] In preferred embodiments, the protein amount of a MybTF
protein in the transgenic plant is increased by at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, or at least 95% or more
in comparison to a wild type plant that is not transformed with the
MybTF nucleic acid.
[0363] More preferably, the transgenic plant, transgenic plant
part, or transgenic plant cell according to the present invention
has been obtained by transformation with a recombinant vector
described herein.
[0364] Suitable methods for transforming or transfecting host cells
including plant cells are well known in the art of plant
biotechnology. Any method may be used to transform the recombinant
expression vector into plant cells to yield the transgenic plants
of the invention. General methods for transforming dicotyledonous
plants are disclosed, for example, in U.S. Pat. Nos. 4,940,838;
5,464,763, and the like. Methods for transforming specific
dicotyledonous plants, for example, cotton, are set forth in U.S.
Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soy transformation
methods are set forth in U.S. Pat. Nos. 4,992,375; 5,416,011;
5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may be used.
Transformation methods may include direct and indirect methods of
transformation. Suitable direct methods include polyethylene glycol
induced DNA uptake, liposome-mediated transformation (U.S. Pat. No.
4,536,475), biolistic methods using the gene gun (Fromm M E et al.,
Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell
2:603, 1990), electroporation, incubation of dry embryos in
DNA-comprising solution, and microinjection. In the case of these
direct transformation methods, the plasmids used need not meet any
particular requirements. Simple plasmids, such as those of the pUC
series, pBR322, M13mp series, pACYC184 and the like can be used. If
intact plants are to be regenerated from the transformed cells, an
additional selectable marker gene is preferably located on the
plasmid. The direct transformation techniques are equally suitable
for dicotyledonous and monocotyledonous plants.
[0365] Transformation can also be carried out by bacterial
infection by means of Agrobacterium (for example EP 0 116 718),
viral infection by means of viral vectors (EP 0 067 553; U.S. Pat.
No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP
0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611). Agrobacterium
based transformation techniques (especially for dicotyledonous
plants) are well known in the art. The Agrobacterium strain (e.g.,
Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a
plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred
to the plant following infection with Agrobacterium. The T-DNA
(transferred DNA) is integrated into the genome of the plant cell.
The T-DNA may be localized on the Ri- or Ti-plasmid or is
separately comprised in a so-called binary vector. Methods for the
Agrobacterium-mediated transformation are described, for example,
in Horsch R B et al. (1985) Science 225:1229. The
Agrobacterium-mediated transformation is best suited to
dicotyledonous plants but has also been adapted to monocotyledonous
plants. The transformation of plants by Agrobacteria is described
in, for example, White F F,
[0366] Vectors for Gene Transfer in Higher Plants, Transgenic
Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung
and R. Wu, Academic Press, 1993, pp. 15-38; Jenes B et al.
Techniques for Gene Transfer, Transgenic Plants, Vol. 1,
Engineering and Utilization, edited by S. D. Kung and R. Wu,
Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev Plant
Physiol Plant Molec Biol 42:205-225. Transformation may result in
transient or stable transformation and expression. Although a
nucleotide sequence of the present invention can be inserted into
any plant and plant cell falling within these broad classes, it is
particularly useful in crop plant cells.
[0367] The genetically modified plant cells can be regenerated via
all methods with which the skilled worker is familiar. Suitable
methods can be found in the abovementioned publications by S. D.
Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0368] After transformation, plant cells or cell groupings may be
selected for the presence of one or more markers which are encoded
by plant-expressible genes co-transferred with the gene of
interest, following which the transformed material is regenerated
into a whole plant. To select transformed plants, the plant
material obtained in the transformation is, as a rule, subjected to
selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Alternatively, the transformed plants are screened for the
presence of a selectable marker such as the ones described above.
The transformed plants may also be directly selected by screening
for the presence of the MybTF nucleic acid.
[0369] Following DNA transfer and regeneration, putatively
transformed plants may also be evaluated, for instance using
Southern analysis, for the presence of the gene of interest, copy
number and/or genomic organisation. Alternatively or additionally,
expression levels of the newly introduced DNA may be monitored
using Northern and/or Western analysis, both techniques being well
known to persons having ordinary skill in the art.
[0370] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed and homozygous second-generation
(or T2) transformants selected, and the T2 plants may then further
be propagated through classical breeding techniques. The generated
transformed organisms may take a variety of forms. For example,
they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain
the expression cassette); grafts of transformed and untransformed
tissues (e.g., in plants, a transformed rootstock grafted to an
untransformed scion).
[0371] Preferably, constructs or vectors or expression cassettes
are not present in the genome of the original plant or are present
in the genome of the transgenic plant not at their natural locus of
the genome of the original plant.
[0372] Preferably, the transgenic plant of the present invention or
the plant obtained by the method of the present invention has
increased resistance against fungal pathogens, preferably rust
pathogens (i.e., fungal pathogens of the order Pucciniales),
preferably against fungal pathogens of the family Phacopsoraceae,
more preferably against fungal pathogens of the genus Phacopsora,
most preferably against Phakopsora pachyrhizi and Phakopsora
meibomice, also known as soybean rust. Preferably, resistance
against Phakopsora pachyrhizi and/or Phakopsora meibomiae is
increased.
[0373] Preferably, the plant, plant part, or plant cell is a plant
or derived from a plant selected from the group consisting of
beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches,
groundnut, rice, wheat, barley, arabidopsis, lentil, banana,
canola, cotton, potatoe, corn, sugar cane, alfalfa, and sugar
beet.
[0374] In one embodiment of the present invention the plant is
selected from the group consisting of beans, soya, pea, clover,
kudzu, lucerne, lentils, lupins, vetches, and/or groundnut.
Preferably, the plant is a legume, comprising plants of the genus
Phaseolus (comprising French bean, dwarf bean, climbing bean
(Phaseolus vulgaris), Lima bean (Phaseolus lunatus L.), Tepary bean
(Phaseolus acutifolius A. Gray), runner bean (Phaseolus
coccineus)); the genus Glycine (comprising Glycine soja, soybeans
(Glycine max (L.) Merill)); pea (Pisum) (comprising shelling peas
(Pisum sativum L. convar. sativum), also called smooth or
roundseeded peas; marrowfat pea (Pisum sativum L. convar. medullare
Alef. emend. C.O. Lehm), sugar pea (Pisum sativum L. convar.
axiphium Alef emend. C.O. Lehm), also called snow pea,
edible-podded pea or mangetout, (Pisum granda sneida L. convar.
sneidulo p. shneiderium)); peanut (Arachis hypogaea), clover
(Trifolium spec.), medick (Medicago), kudzu vine (Pueraria lobata),
common lucerne, alfalfa (M. sativa L.), chickpea (Cicer), lentils
(Lens) (Lens culinaris Medik.), lupins (Lupinus); vetches (Vicia),
field bean, broad bean (Vicia faba), vetchling (Lathyrus)
(comprising chickling pea (Lathyrus sativus), heath pea (Lathyrus
tuberosus)); genus Vigna (comprising moth bean (Vigna aconitifolia
(Jacq.) Marechal), adzuki bean (Vigna angularis (Willd.) Ohwi &
H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mung bean (Vigna
radiata (L.) R. Wilczek), bambara groundnut (Vigna subterrane (L.)
Verdc.), rice bean (Vigna umbellata (Thunb.) Ohwi & H. Ohashi),
Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., in the
three subspecies asparagus bean, cowpea, catjang bean)); pigeonpea
(Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising
geocarpa groundnut (Macrotyloma geocarpum (Harms) Marechal &
Baudet), horse bean (Macrotyloma uniflorum (Lam.) Verdc.); goa bean
(Psophocarpus tetragonolobus (L.) DC.), African yam bean
(Sphenostylis stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian
black bean, dolichos bean, lablab bean (Lablab purpureus (L.)
Sweet), yam bean (Pachyrhizus), guar bean (Cyamopsis tetragonolobus
(L.) Taub.); and/or the genus Canavalia (comprising jack bean
(Canavalia ensiformis (L.) DC.), sword bean (Canavalia gladiata
(Jacq.) DC.).
[0375] Further preferred is a plant selected from the group
consisting of beans, soya, pea, clover, kudzu, lucerne, lentils,
lupins, vetches, and groundnut. Most preferably, the plant, plant
part, or plant cell is or is derived from soy.
[0376] Preferably, the transgenic plant of the present invention or
the plant obtained by the method of the present invention is a
soybean plant and has increased resistance against fungal pathogens
of the order Pucciniales (rust), preferably, of the family
Phacopsoraceae, more preferably against fungal pathogens of the
genus Phacopsora, most preferably against Phakopsora pachyrhizi and
Phakopsora meibomiae, also known as soybean rust. Preferably,
resistance against Phakopsora pachyrhizi and/or Phakopsora
meibomiae is increased.
[0377] Methods for the Production of Transgenic Plants
[0378] One embodiment according to the present invention provides a
method for producing a transgenic plant, a transgenic plant part,
or a transgenic plant cell resistant to a fungal pathogen,
preferably of the family Phacosporaceae, for example soybean rust,
wherein the recombinant nucleic acid used to generate a transgenic
plant comprises a promoter that is functional in the plant cell,
operably linked to a MybTF nucleic acid, which is preferably SEQ ID
NO: 2, 3, 6, 4, or 1, and a terminator regulatory sequence.
[0379] In one embodiment, the present invention refers to a method
for the production of a transgenic plant, transgenic plant part, or
transgenic plant cell having increased fungal resistance,
comprising [0380] (a) introducing a recombinant vector construct
according to the present invention into a plant, a plant part or a
plant cell and [0381] (b) generating a transgenic plant from the
plant, plant part or plant cell.
[0382] Preferably, the method for the production of the transgenic
plant, transgenic plant part, or transgenic plant cell further
comprises the step [0383] (c) expressing the MybTF protein,
preferably encoded by a nucleic acid comprising [0384] (i) an
exogenous nucleic acid having at least 70% identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55, a functional fragment thereof, an orthologue
or a paralogue thereof, or a splice variant thereof; [0385] (ii) an
exogenous nucleic acid encoding a protein comprising an amino acid
sequence having at least 70% identity with SEQ ID NO: 7, 5, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, or a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0386] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecute codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0387] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code.
[0388] Preferably, said introducing and expressing does not
comprise an essentially biological process.
[0389] More preferably, the method for the production of the
transgenic plant, transgenic plant part, or transgenic plant cell
further comprises the step [0390] (c) expressing the MybTF protein,
preferably encoded by [0391] (i) an exogenous nucleic acid having
at least 70% identity with SEQ ID NO: 1, a functional fragment
thereof, an orthologue or a paralogue thereof, or a splice variant
thereof; [0392] (ii) an exogenous nucleic acid encoding a protein
having at least 70% identity with SEQ ID NO: 7 or 5, or a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0393] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0394] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code.
[0395] More preferably, the method for the production of the
transgenic plant, transgenic plant part, or transgenic plant cell
further comprises the step [0396] (c) expressing the MybTF protein,
preferably encoded by [0397] (i) an exogenous nucleic acid having
at least 70% identity with SEQ ID NO: 2 or 6, a functional fragment
thereof, an orthologue or a paralogue thereof, or a splice variant
thereof; [0398] (ii) an exogenous nucleic acid encoding a protein
having at least 70% identity with SEQ ID NO: 7, or a functional
fragment thereof, an orthologue or a paralogue thereof; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; [0399] (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; and/or by [0400] (iv) an exogenous
nucleic acid encoding the same MybTF protein as the nucleic acids
of (i) to (iii) above, but differing from the nucleic acids of (i)
to (iii) above due to the degeneracy of the genetic code.
[0401] More preferably, the method for the production of the
transgenic plant, transgenic plant part, or transgenic plant cell
further comprises the step [0402] (c) expressing the MybTF protein,
preferably encoded by [0403] (i) an exogenous nucleic acid having
at least 70% identity with SEQ ID NO: 3 or 4, a functional fragment
thereof, an orthologue or a paralogue thereof, or a splice variant
thereof; [0404] (ii) an exogenous nucleic acid encoding a protein
having at least 70% identity with SEQ ID NO: 5, or a functional
fragment thereof, an orthologue or a paralogue thereof; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; [0405] (iii) an exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 5;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; and/or by [0406] (iv) an exogenous
nucleic acid encoding the same MybTF protein as the nucleic acids
of (i) to (iii) above, but differing from the nucleic acids of (i)
to (iii) above due to the degeneracy of the genetic code.
[0407] Preferably, the method for the production of the transgenic
plant, transgenic plant part, or transgenic plant cell further
comprises the step of selecting a transgenic plant expressing
[0408] (i) an exogenous nucleic acid having at least 70% sequence
identity, at least 80%, at least 90%, at least 95%, at least 98%,
at least 99% sequence identity, or even 100% sequence identity with
SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, or 55, or a functional fragment
thereof, or an orthologue or a paralogue thereof, or a splice
variant thereof; [0409] (ii) an exogenous nucleic acid coding for a
protein having at least 70% sequence identity, at least 80%, at
least 90%, at least 95%, at least 98%, at least 99% sequence
identity, or even 100% sequence identity with SEQ ID NO: 7, 5, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0410] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or [0411] (iv) an
exogenous nucleic acid encoding the same MybTF polypeptide as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code.
[0412] Preferably, the method for the production of the transgenic
plant, transgenic plant part, or transgenic plant cell additionally
comprises the step of harvesting the seeds of the transgenic plant
and planting the seeds and growing the seeds to plants, wherein the
grown plant(s) comprises [0413] (i) the exogenous nucleic acid
having at least 70% identity with SEQ ID NO: 2, 3, 1, 6, 4, 8,
9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
or 55, a functional fragment thereof, an orthologue or a paralogue
thereof, or a splice variant thereof; [0414] (ii) the exogenous
nucleic acid encoding a protein comprising an amino acid sequence
having at least 70% identity with SEQ ID NO: 7, 5, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, or a functional
fragment thereof, an orthologue or a paralogue thereof; preferably
the encoded protein confers enhanced fungal resistance relative to
control plants; [0415] (iii) the exogenous nucleic acid capable of
hybridizing under stringent conditions with a complementary
sequence of any of the nucleic acids according to (i) or (ii);
preferably encoding a MybTF protein; preferably wherein the nucleic
acid molecule codes for a polypeptide which has essentially
identical properties to the polypeptide described in SEQ ID NO: 7
or 5; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; and/or [0416] (iv) the
exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code; preferably, the step of harvesting the seeds of the
transgenic plant and planting the seeds and growing the seeds to
plants, wherein the grown plant(s) comprises [0417] (i) the
exogenous nucleic acid having at least 70% identity with SEQ ID NO:
2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, or 55, a functional fragment thereof, an orthologue
or a paralogue thereof, or a splice variant thereof; [0418] (ii)
the exogenous nucleic acid encoding a protein comprising an amino
acid sequence having at least 70% identity with SEQ ID NO: 7, 5,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56,
or a functional fragment thereof, an orthologue or a paralogue
thereof; preferably the encoded protein confers enhanced fungal
resistance relative to control plants; [0419] (iii) the exogenous
nucleic acid capable of hybridizing under stringent conditions with
a complementary sequence of any of the nucleic acids according to
(i) or (ii); preferably encoding a MybTF protein; preferably
wherein the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or [0420] (iv)
the exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code; is repeated more than one time, preferably, 1, 2, 3, 4, 5, 6,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.
[0421] The transgenic plants may be selected by known methods as
described above (e.g., by screening for the presence of one or more
markers which are encoded by plant-expressible genes co-transferred
with the MybTF gene or by directly screening for the MybTF nucleic
acid).
[0422] Furthermore, the use of the exogenous MybTF nucleic acid or
the recombinant vector construct comprising the MybTF nucleic acid
for the transformation of a plant, plant part, or plant cell to
provide a fungal resistant plant, plant part, or plant cell is
provided.
[0423] Harvestable Parts and Products
[0424] Harvestable parts of the transgenic plant according to the
present invention are part of the invention. Preferably, the
harvestable parts comprise the MybTF nucleic acid or MybTF protein.
The harvestable parts may be seeds, roots, leaves and/or flowers
comprising the MybTF nucleic acid or MybTF protein or parts
thereof. Preferred parts of soy plants are soy beans comprising the
MybTF nucleic acid or MybTF protein.
[0425] Products derived from a transgenic plant according to the
present invention, parts thereof or harvestable parts thereof are
part of the invention. A preferred product is meal or oil,
preferably, soybean meal or soybean oil. Preferably, the soybean
meal and/or oil comprises the MybTF nucleic acid or MybTF
protein.
[0426] Preferably the harvestable parts of the transgenic plant
according to the present invention or the products derived from a
transgenic plant comprise an exogenous nucleic acid molecule
consisting of or comprising a nucleic acid selected from the group
consisting of: [0427] (i) an exogenous nucleic acid having in
increasing order of preference at least at least 70%, at least 71%,
at least 72%, at least 73%, at least 74%, at least 75%, at least
76%, at least 77%, at least 78%, at least 79%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity to the nucleic acid sequence represented
by SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, or 55, or a functional fragment,
derivative, orthologue, or paralogue thereof, or a splice variant
thereof; [0428] (ii) an exogenous nucleic acid encoding a MybTF
protein comprising an amino acid sequence having in increasing
order of preference at least 70%, at least 71%, at least 72%, at
least 73%, at least 74%, at least 75%, at least 76%, at least 77%,
at least 78%, at least 79%, at least 80%, at least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence
identity to the amino acid sequence represented by SEQ ID NO: 7, 5,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56,
or a functional fragment, derivative, orthologue, or paralogue
thereof; preferably the MybTF protein has essentially the same
biological activity as a MybTF protein encoded by SEQ ID NO: 1, 2,
4, or 6; preferably the MybTF protein confers enhanced fungal
resistance relative to control plants; [0429] (iii) an exogenous
nucleic acid molecule which hybridizes with a complementary
sequence of any of the nucleic acid molecules of (i) or (ii) under
high stringency hybridization conditions; preferably encoding a
MybTF protein; preferably wherein the nucleic acid molecule codes
for a polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0430] (iv) an exogenous nucleic acid encoding the same
MybTF protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code, or wherein the harvestable part
of the transgenic plant or the product derived from the transgenic
plant comprises a MybTF protein encoded by any one of the MybTF
nucleic acids of (i) to (iv).
[0431] Methods for Manufacturing a Product
[0432] In one embodiment the method for the production of a product
comprises [0433] a) growing the plants of the invention or
obtainable by the methods of invention and [0434] b) producing said
product from or by the plants of the invention and/or parts, e.g.
seeds, of these plants.
[0435] In a further embodiment the method comprises the steps a)
growing the plants of the invention, b) removing the harvestable
parts as defined above from the plants and c) producing said
product from or by the harvestable parts of the invention.
[0436] Preferably the products obtained by said method comprises an
exogenous nucleic acid molecule consisting of or comprising a
nucleic acid selected from the group consisting of: [0437] (i) an
exogenous nucleic acid having in increasing order of preference at
least 70%, at least 71%, at least 72%, at least 73%, at least 74%,
at least 75%, at least 76%, at least 77%, at least 78%, at least
79%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% or 100% sequence identity to the nucleic
acid sequence represented by SEQ ID NO: 2, 3, 1, 6, 4, 8, 9-24, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55, or a
functional fragment, derivative, orthologue, or paralogue thereof,
or a splice variant thereof; [0438] (ii) an exogenous nucleic acid
encoding a MybTF protein comprising an amino acid sequence having
in increasing order of preference at least 70%, at least 71%, at
least 72%, at least 73%, at least 74%, at least 75%, at least 76%,
at least 77%, at least 78%, at least 79%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to the amino acid sequence represented by SEQ ID
NO: 7, 5, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, or 56, or a functional fragment, derivative, orthologue, or
paralogue thereof; preferably the MybTF protein has essentially the
same biological activity as a MybTF protein encoded by SEQ ID NO:
1, 2, 4, or 6; preferably the MybTF protein confers enhanced fungal
resistance relative to control plants; [0439] (iii) an exogenous
nucleic acid molecule which hybridizes with a complementary
sequence of any of the nucleic acid molecules of (i) or (ii) under
high stringency hybridization conditions; preferably encoding a
MybTF protein; preferably wherein the nucleic acid molecule codes
for a polypeptide which has essentially identical properties to the
polypeptide described in SEQ ID NO: 7 or 5; preferably the encoded
protein confers enhanced fungal resistance relative to control
plants; and [0440] (iv) an exogenous nucleic acid encoding the same
MybTF protein as the MybTF nucleic acids of (i) to (iii) above, but
differing from the MybTF nucleic acids of (i) to (iii) above due to
the degeneracy of the genetic code, or wherein the product obtained
by said method comprises a MybTF protein encoded by any one of the
MybTF nucleic acids of (i) to (iv).
[0441] The product may be produced at the site where the plant has
been grown, the plants and/or parts thereof may be removed from the
site where the plants have been grown to produce the product.
Typically, the plant is grown, the desired harvestable parts are
removed from the plant, if feasible in repeated cycles, and the
product made from the harvestable parts of the plant. The step of
growing the plant may be performed only once each time the methods
of the invention is performed, while allowing repeated times the
steps of product production e.g. by repeated removal of harvestable
parts of the plants of the invention and if necessary further
processing of these parts to arrive at the product. It is also
possible that the step of growing the plants of the invention is
repeated and plants or harvestable parts are stored until the
production of the product is then performed once for the
accumulated plants or plant parts. Also, the steps of growing the
plants and producing the product may be performed with an overlap
in time, even simultaneously to a large extend or sequentially.
Generally the plants are grown for some time before the product is
produced.
[0442] In one embodiment the products produced by said methods of
the invention are plant products such as, but not limited to, a
foodstuff, feedstuff, a food supplement, feed supplement, fiber,
cosmetic and/or pharmaceutical. Foodstuffs are regarded as
compositions used for nutrition and/or for supplementing nutrition.
Animal feedstuffs and animal feed supplements, in particular, are
regarded as foodstuffs.
[0443] In another embodiment the inventive methods for the
production are used to make agricultural products such as, but not
limited to, plant extracts, proteins, amino acids, carbohydrates,
fats, oils, polymers, vitamins, and the like.
[0444] It is possible that a plant product consists of one or more
agricultural products to a large extent.
[0445] Methods for Breeding/Methods for Plant Improvement/Methods
Plant Variety Production
[0446] The transgenic plants of the invention may be crossed with
similar transgenic plants or with transgenic plants lacking the
nucleic acids of the invention or with non-transgenic plants, using
known methods of plant breeding, to prepare seeds. Further, the
transgenic plant cells or plants of the present invention may
comprise, and/or be crossed to another transgenic plant that
comprises one or more exogenous nucleic acids, thus creating a
"stack" of transgenes in the plant and/or its progeny. The seed is
then planted to obtain a crossed fertile transgenic plant
comprising the MybTF nucleic acid. The crossed fertile transgenic
plant may have the particular expression cassette inherited through
a female parent or through a male parent. The second plant may be
an inbred plant. The crossed fertile transgenic may be a hybrid.
Also included within the present invention are seeds of any of
these crossed fertile transgenic plants. The seeds of this
invention can be harvested from fertile transgenic plants and be
used to grow progeny generations of transformed plants of this
invention including hybrid plant lines comprising the exogenous
nucleic acid.
[0447] Thus, one embodiment of the present invention is a method
for breeding a fungal resistant plant comprising the steps of
[0448] (a) crossing a transgenic plant described herein or a plant
obtainable by a method described herein with a second plant; [0449]
(b) obtaining a seed or seeds resulting from the crossing step
described in (a); [0450] (c) planting said seed or seeds and
growing the seed or seeds to plants; and [0451] (d) selecting from
said plants the plants expressing a MybTF protein, preferably
encoded by a nucleic acid comprising [0452] (i) an exogenous
nucleic acid having at least 70% identity with SEQ ID NO: 2, 3, 1,
6, 4, 8, 9-24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, or 55, a functional fragment thereof, an orthologue or a
paralogue thereof, or a splice variant thereof; [0453] (ii) an
exogenous nucleic acid encoding a protein comprising an amino acid
sequence having at least 70% identity with SEQ ID NO: 7, 5, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, or a
functional fragment thereof, an orthologue or a paralogue thereof;
preferably the encoded protein confers enhanced fungal resistance
relative to control plants; [0454] (iii) an exogenous nucleic acid
capable of hybridizing under stringent conditions with a
complementary sequence of any of the nucleic acids according to (i)
or (ii); preferably encoding a MybTF protein; preferably wherein
the nucleic acid molecule codes for a polypeptide which has
essentially identical properties to the polypeptide described in
SEQ ID NO: 7 or 5; preferably the encoded protein confers enhanced
fungal resistance relative to control plants; and/or by [0455] (iv)
an exogenous nucleic acid encoding the same MybTF protein as the
nucleic acids of (i) to (iii) above, but differing from the nucleic
acids of (i) to (iii) above due to the degeneracy of the genetic
code.
[0456] Another preferred embodiment is a method for plant
improvement comprising [0457] (a) obtaining a transgenic plant by
any of the methods of the present invention; [0458] (b) combining
within one plant cell the genetic material of at least one plant
cell of the plant of (a) with the genetic material of at least one
cell differing in one or more gene from the plant cells of the
plants of (a) or crossing the transgenic plant of (a) with a second
plant; [0459] (c) obtaining seed from at least one plant generated
from the one plant cell of (b) or the plant of the cross of step
(b); [0460] (d) planting said seeds and growing the seeds to
plants; and [0461] (e) selecting from said plants, plants
expressing the nucleic acid encoding the MybTF protein; and
optionally [0462] (f) producing propagation material from the
plants expressing the nucleic acid encoding the MybTF protein.
[0463] The transgenic plants may be selected by known methods as
described above (e.g., by screening for the presence of one or more
markers which are encoded by plant-expressible genes co-transferred
with the MybTF gene or screening for the MybTF nucleic acid
itself).
[0464] According to the present invention, the introduced MybTF
nucleic acid may be maintained in the plant cell stably if it is
incorporated into a non-chromosomal autonomous replicon or
integrated into the plant chromosomes. Whether present in an
extra-chromosomal nonreplicating or replicating vector construct or
a vector construct that is integrated into a chromosome, the
exogenous MybTF nucleic acid preferably resides in a plant
expression cassette. A plant expression cassette preferably
contains regulatory sequences capable of driving gene expression in
plant cells that are functional linked so that each sequence can
fulfill its function, for example, termination of transcription by
polyadenylation signals. Preferred polyadenylation signals are
those originating from Agrobacterium tumefaciens t-DNA such as the
gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen
et al., 1984, EMBO J. 3:835) or functional equivalents thereof, but
also all other terminators functionally active in plants are
suitable. As plant gene expression is very often not limited on
transcriptional levels, a plant expression cassette preferably
contains other functional linked sequences like translational
enhancers such as the overdrive-sequence containing the
5'-untranslated leader sequence from tobacco mosaic virus
increasing the polypeptide per RNA ratio (Gallie et al., 1987,
Nucl. Acids Research 15:8693-8711). Examples of plant expression
vectors include those detailed in: Becker, D. et al., 1992, New
plant binary vectors with selectable markers located proximal to
the left border, Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984,
Binary Agrobacterium vectors for plant transformation, Nucl. Acid.
Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants;
in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.:
Kung and R. Wu, Academic Press, 1993, S. 15-38.
EXAMPLES
[0465] The following examples are not intended to limit the scope
of the claims to the invention, but are rather intended to be
exemplary of certain embodiments. Any variations in the exemplified
methods that occur to the skilled artisan are intended to fall
within the scope of the present invention.
Example 1
General Methods
[0466] The chemical synthesis of oligonucleotides can be affected,
for example, in the known fashion using the phosphoamidite method
(Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The
cloning steps carried out for the purposes of the present invention
such as, for example, restriction cleavages, agarose gel
electrophoresis, purification of DNA fragments, transfer of nucleic
acids to nitrocellulose and nylon membranes, linking DNA fragments,
transformation of E. coli cells, bacterial cultures, phage
multiplication and sequence analysis of recombinant DNA, are
carried out as described by Sambrook et al. Cold Spring Harbor
Laboratory Press (1989), ISBN 0-87969-309-6. The sequencing of
recombinant DNA molecules is carried out with an MWG-Licor laser
fluorescence DNA sequencer following the method of Sanger (Sanger
et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).
Example 2
Cloning of Overexpression Vector Constructs
[0467] The genomic DNA sequence and the optimized cDNAs of the
MybTF gene mentioned in this application were generated by DNA
synthesis (Geneart, Regensburg, Germany).
[0468] The MybTF DNA (as shown in SEQ ID NO: 1) was synthesized in
a way that a Pacl restriction site is located in front of the
start-ATG and a Ascl restriction site downstream of the stop-codon.
The synthesized DNA was digested using the restriction enzymes Pacl
and Ascl (NEB Biolabs) and ligated in a Pacl/Ascl digested Gateway
pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, Calif.,
USA) in a way that the full-length fragment is located in sense
direction between the parsley ubiquitin promoter and the
Agrobacterium tumefaciens derived octopine synthase terminator
(t-OCS). The PcUbi promoter regulates constitutive expression of
the ubi4-2 gene (accession number X64345) of Petroselinum crispum
(Kawalleck et al. 1993 Plant Molecular Biology 21(4): 673-684).
[0469] To obtain the binary plant transformation vector, a triple
LR reaction (Gateway system, Invitrogen, Life Technologies,
Carlsbad, Calif., USA) was performed according to manufacturer's
protocol by using an empty pENTRY-A vector, the PcUbi
promoter::MybTF::OCSterminator in the above described pENTRY-B
vector and an empty pENTRY-C. As target a binary pDEST vector was
used which is composed of: (1) a Spectinomycin/Streptomycin
resistance cassette for bacterial selection (2) a pVS1 origin for
replication in Agrobacteria (3) a ColE1 origin of replication for
stable maintenance in E. coli and (4) between the right and left
border an AHAS selection under control of a PcUbi-promoter (see
FIG. 2). The recombination reaction was transformed into E. coli
(DH5alpha), mini-prepped and screened by specific restriction
digestions. A positive clone from each vector construct was
sequenced and submitted soy transformation.
Example 3
Soy Transformation
[0470] The expression vector constructs (see example 2) were
transformed into soy.
[0471] 3.1 Sterilization and Germination of Soy Seeds
[0472] Virtually any seed of any soy variety can be employed in the
method of the invention. A variety of soybean cultivar (including
Jack, Williams 82, Jake, Stoddard and Resnik) is appropriate for
soy transformation. Soy seeds were sterilized in a chamber with a
chlorine gas produced by adding 3.5 ml 12N HCl drop wise into 100
ml bleach (5.25% sodium hypochlorite) in a desiccator with a
tightly fitting lid. After 24 to 48 hours in the chamber, seeds
were removed and approximately 18 to 20 seeds were plated on solid
GM medium with or without 5 .mu.M 6-benzyl-aminopurine (BAP) in 100
mm Petri dishes. Seedlings without BAP are more elongated and roots
develop, especially secondary and lateral root formation. BAP
strengthens the seedling by forming a shorter and stockier
seedling.
[0473] Seven-day-old seedlings grown in the light (>100
.mu.Einstein/m.sup.2s) at 25.degree. C. were used for explant
material for the three-explant types. At this time, the seed coat
was split, and the epicotyl with the unifoliate leaves have grown
to, at minimum, the length of the cotyledons. The epicotyl should
be at least 0.5 cm to avoid the cotyledonary-node tissue (since
soycultivars and seed lots may vary in the developmental time a
description of the germination stage is more accurate than a
specific germination time).
[0474] For inoculation of entire seedlings, see Method A (example
3.3.1 and 3.3.2) or leaf explants, see Method B (example
3.3.3).
[0475] For Method C (see example 3.3.4), the hypocotyl and one and
a half or part of both cotyledons were removed from each seedling.
The seedlings were then placed on propagation media for 2 to 4
weeks. The seedlings produce several branched shoots to obtain
explants from. The majority of the explants originated from the
plantlet growing from the apical bud.
[0476] These explants were preferably used as target tissue.
[0477] 3.2--Growth and Preparation of Agrobacterium Culture
[0478] Agrobacterium cultures were prepared by streaking
Agrobacterium (e.g., A. tumefaciens or A. rhizogenes) carrying the
desired binary vector (e.g. H. Klee. R. Horsch and S. Rogers 1987
Agrobacterium-Mediated Plant Transformation and its further
Applications to Plant Biology; Annual Review of Plant Physiology
Vol. 38: 467-486) onto solid YEP growth medium (YEP media: 10 g
yeast extract, 10 g Bacto Peptone, 5 g NaCl, Adjust pH to 7.0, and
bring final volume to 1 liter with H2O, for YEP agar plates add 20
g Agar, autoclave) and incubating at 25.degree. C. until colonies
appeared (about 2 days). Depending on the selectable marker genes
present on the Ti or Ri plasmid, the binary vector, and the
bacterial chromosomes, different selection compounds were be used
for A. tumefaciens and A. rhizogenes selection in the YEP solid and
liquid media. Various Agrobacterium strains can be used for the
transformation method.
[0479] After approximately two days, a single colony (with a
sterile toothpick) was picked and 50 ml of liquid YEP was
inoculated with antibiotics and shaken at 175 rpm (25.degree. C.)
until an OD600 between 0.8-1.0 is reached (approximately 2 d).
Working glycerol stocks (15%) for transformation are prepared and
one-ml of Agrobacterium stock aliquoted into 1.5 ml Eppendorf tubes
then stored at -80.degree. C.
[0480] The day before explant inoculation, 200 ml of YEP were
inoculated with 5 .mu.l to 3 ml of working Agrobacterium stock in a
500 ml Erlenmeyer flask. The flask was shaken overnight at
25.degree. C. until the OD600 was between 0.8 and 1.0. Before
preparing the soy explants, the Agrobacteria were pelleted by
centrifugation for 10 min at 5,500.times.g at 20.degree. C. The
pellet was resuspended in liquid CCM to the desired density (OD600
0.5-0.8) and placed at room temperature at least 30 min before
use.
[0481] 3.3--Explant Preparation and Co-Cultivation (Inoculation)
3.3.1 Method A: Explant Preparation on the Day of
Transformation.
[0482] Seedlings at this time had elongated epicotyls from at least
0.5 cm but generally between 0.5 and 2 cm. Elongated epicotyls up
to 4 cm in length had been successfully employed.
[0483] Explants were then prepared with: i) with or without some
roots, ii) with a partial, one or both cotyledons, all preformed
leaves were removed including apical meristem, and the node located
at the first set of leaves was injured with several cuts using a
sharp scalpel.
[0484] This cutting at the node not only induced Agrobacterium
infection but also distributed the axillary meristem cells and
damaged pre-formed shoots. After wounding and preparation, the
explants were set aside in a Petri dish and subsequently
co-cultivated with the liquid CCM/Agrobacterium mixture for 30
minutes. The explants were then removed from the liquid medium and
plated on top of a sterile filter paper on 15.times.100 mm Petri
plates with solid co-cultivation medium. The wounded target tissues
were placed such that they are in direct contact with the
medium.
[0485] 3.3.2 Modified Method A: Epicotyl Explant Preparation
[0486] Soyepicotyl segments prepared from 4 to 8 d old seedlings
were used as explants for regeneration and transformation. Seeds of
soya cv. L00106CN, 93-41131 and Jack were germinated in 1/10 MS
salts or a similar composition medium with or without cytokinins
for 4 to 8 d. Epicotyl explants were prepared by removing the
cotyledonary node and stem node from the stem section. The epicotyl
was cut into 2 to 5 segments. Especially preferred are segments
attached to the primary or higher node comprising axillary
meristematic tissue.
[0487] The explants were used for Agrobacterium infection.
Agrobacterium AGL1 harboring a plasmid with the gene of interest
(GOI) and the AHAS, bar or dsdA selectable marker gene was cultured
in LB medium with appropriate antibiotics overnight, harvested and
resuspended in a inoculation medium with acetosyringone. Freshly
prepared epicotyl segments were soaked in the Agrobacterium
suspension for 30 to 60 min and then the explants were blotted dry
on sterile filter papers. The inoculated explants were then
cultured on a coculture medium with L-cysteine and TTD and other
chemicals such as acetosyringone for increasing T-DNA delivery for
2 to 4 d. The infected epicotyl explants were then placed on a
shoot induction medium with selection agents such as imazapyr (for
AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA
gene). The regenerated shoots were subcultured on elongation medium
with the selective agent.
[0488] For regeneration of transgenic plants the segments were then
cultured on a medium with cytokinins such as BAP, TDZ and/or
Kinetin for shoot induction. After 4 to 8 weeks, the cultured
tissues were transferred to a medium with lower concentration of
cytokinin for shoot elongation. Elongated shoots were transferred
to a medium with auxin for rooting and plant development. Multiple
shoots were regenerated.
[0489] Many stable transformed sectors showing strong cDNA
expression were recovered. Soyplants were regenerated from epicotyl
explants. Efficient T-DNA delivery and stable transformed sectors
were demonstrated.
[0490] 3.3.3 Method B: Leaf Explants
[0491] For the preparation of the leaf explant the cotyledon was
removed from the hypocotyl. The cotyledons were separated from one
another and the epicotyl is removed. The primary leaves, which
consist of the lamina, the petiole, and the stipules, were removed
from the epicotyl by carefully cutting at the base of the stipules
such that the axillary meristems were included on the explant. To
wound the explant as well as to stimulate de novo shoot formation,
any pre-formed shoots were removed and the area between the
stipules was cut with a sharp scalpel 3 to 5 times.
[0492] The explants are either completely immersed or the wounded
petiole end dipped into the Agrobacterium suspension immediately
after explant preparation. After inoculation, the explants are
blotted onto sterile filter paper to remove excess Agrobacterium
culture and place explants with the wounded side in contact with a
round 7 cm Whatman paper overlaying the solid CCM medium (see
above). This filter paper prevents A. tumefaciens overgrowth on the
soy-explants. Wrap five plates with Parafilm.TM. "M" (American
National Can, Chicago, III., USA) and incubate for three to five
days in the dark or light at 25.degree. C.
[0493] 3.3.4 Method C: Propagated Axillary Meristem
[0494] For the preparation of the propagated axillary meristem
explant propagated 3-4 week-old plantlets were used. Axillary
meristem explants can be pre-pared from the first to the fourth
node. An average of three to four explants could be obtained from
each seedling. The explants were prepared from plantlets by cutting
0.5 to 1.0 cm below the axillary node on the internode and removing
the petiole and leaf from the explant. The tip where the axillary
meristems lie was cut with a scalpel to induce de novo shoot growth
and allow access of target cells to the Agrobacterium. Therefore, a
0.5 cm explant included the stem and a bud.
[0495] Once cut, the explants were immediately placed in the
Agrobacterium suspension for 20 to 30 minutes. After inoculation,
the explants were blotted onto sterile filter paper to remove
excess Agrobacterium culture then placed almost completely immersed
in solid CCM or on top of a round 7 cm filter paper overlaying the
solid CCM, depending on the Agrobacterium strain. This filter paper
prevents Agrobacterium overgrowth on the soy-explants. Plates were
wrapped with Parafilm.TM. "M" (American National Can, Chicago,
Ill., USA) and incubated for two to three days in the dark at
25.degree. C.
[0496] 3.4--Shoot Induction
[0497] After 3 to 5 days co-cultivation in the dark at 25.degree.
C., the explants were rinsed in liquid SIM medium (to remove excess
Agrobacterium) (SIM, see Olhoft et al 2007 A novel Agrobacterium
rhizogenes-mediated transformation method of soy using primary-node
explants from seedlings In Vitro Cell. Dev. Biol. Plant (2007)
43:536-549; to remove excess Agrobacterium) or Modwash medium
(1.times.B5 major salts, 1.times.B5 minor salts, 1.times.MSIII
iron, 3% Sucrose, 1.times.B5 vitamins, 30 mM MES, 350 mg/L
Timentin.TM. pH 5.6, WO 2005/121345) and blotted dry on sterile
filter paper (to prevent damage especially on the lamina) before
placing on the solid SIM medium. The approximately 5 explants
(Method A) or 10 to 20 (Methods B and C) explants were placed such
that the target tissue was in direct contact with the medium.
During the first 2 weeks, the explants could be cultured with or
without selective medium. Preferably, explants were transferred
onto SIM without selection for one week.
[0498] For leaf explants (Method B), the explant should be placed
into the medium such that it is perpendicular to the surface of the
medium with the petiole imbedded into the medium and the lamina out
of the medium.
[0499] For propagated axillary meristem (Method C), the explant was
placed into the medium such that it was parallel to the surface of
the medium (basipetal) with the explant partially embedded into the
medium.
[0500] Wrap plates with Scotch 394 venting tape (3M, St. Paul,
Minn., USA) were placed in a growth chamber for two weeks with a
temperature averaging 25.degree. C. under 18 h light/6 h dark cycle
at 70-100 .mu.E/m.sup.2s. The explants remained on the SIM medium
with or without selection until de novo shoot growth occurred at
the target area (e.g., axillary meristems at the first node above
the epicotyl). Transfers to fresh medium can occur during this
time. Explants were transferred from the SIM with or without
selection to SIM with selection after about one week. At this time,
there was considerable de novo shoot development at the base of the
petiole of the leaf explants in a variety of SIM (Method B), at the
primary node for seedling explants (Method A), and at the axillary
nodes of propagated explants (Method C).
[0501] Preferably, all shoots formed before transformation were
removed up to 2 weeks after cocultivation to stimulate new growth
from the meristems. This helped to reduce chimerism in the primary
transformant and increase amplification of transgenic meristematic
cells. During this time the explant may or may not be cut into
smaller pieces (i.e. detaching the node from the explant by cutting
the epicotyl).
[0502] 3.5--Shoot Elongation
[0503] After 2 to 4 weeks (or until a mass of shoots was formed) on
SIM medium (preferably with selection), the explants were
transferred to SEM medium (shoot elongation medium, see Olhoft et
al 2007 A novel Agrobacterium rhizogenes-mediated transformation
method of soy using primary-node explants from seedlings. In Vitro
Cell. Dev. Biol. Plant (2007) 43:536-549) that stimulates shoot
elongation of the shoot primordia. This medium may or may not
contain a selection compound.
[0504] After every 2 to 3 weeks, the explants were transferred to
fresh SEM medium (preferably containing selection) after carefully
removing dead tissue. The explants should hold together and not
fragment into pieces and retain somewhat healthy. The explants were
continued to be transferred until the explant dies or shoots
elongate. Elongated shoots >3 cm were removed and placed into RM
medium for about 1 week (Method A and B), or about 2 to 4 weeks
depending on the cultivar (Method C) at which time roots began to
form. In the case of explants with roots, they were transferred
directly into soil. Rooted shoots were transferred to soil and
hardened in a growth chamber for 2 to 3 weeks before transferring
to the greenhouse. Regenerated plants obtained using this method
were fertile and produced on average 500 seeds per plant.
[0505] After 5 days of co-cultivation with Agrobacterium
tumefaciens transient expression of the gene of interest (GOI) was
widespread on the seedling axillary meristem explants especially in
the regions wounding during explant preparation (Method A).
Explants were placed into shoot induction medium without selection
to see how the primary-node responds to shoot induction and
regeneration. Thus far, greater than 70% of the explants were
formed new shoots at this region. Expression of the GOI was stable
after 14 days on SIM, implying integration of the T-DNA into the
soy genome. In addition, preliminary experiments resulted in the
formation of cDNA expressing shoots forming after 3 weeks on
SIM.
[0506] For Method C, the average regeneration time of a soy
plantlet using the propagated axillary meristem protocol was 14
weeks from explant inoculation. Therefore, this method has a quick
regeneration time that leads to fertile, healthy soy plants.
Example 4
Pathogen Assay
[0507] 4.1. Growth of Plants
[0508] 10 T.sub.1 plants per event were potted and grown for 3-4
weeks in the phytochamber (16 h-day- and 8 h-night-Rhythm at a
temperature of 16 and 22.degree. C. and a humidity of 75%) till the
first 2 trifoliate leaves were fully expanded.
[0509] 4.2 Inoculation
[0510] The plants were inoculated with spores of P. pachyrhizi.
[0511] In order to obtain appropriate spore material for the
inoculation, soybean leaves which had been infected with rust 15-20
days ago, were taken 2-3 days before the inoculation and
transferred to agar plates (1% agar in H2O). The leaves were placed
with their upper side onto the agar, which allowed the fungus to
grow through the tissue and to produce very young spores. For the
inoculation solution, the spores were knocked off the leaves and
were added to a Tween-H2O solution. The counting of spores was
performed under a light microscope by means of a Thoma counting
chamber. For the inoculation of the plants, the spore suspension
was added into a compressed-air operated spray flask and applied
uniformly onto the plants or the leaves until the leaf surface is
well moisturized. For macroscopic assays we used a spore density of
1-5.times.10.sup.5 spores/ml. For the microscopy, a density of
>5.times.10.sup.5 spores/ml is used. The inoculated plants were
placed for 24 hours in a greenhouse chamber with an average of
22.degree. C. and >90% of air humidity. The following
cultivation was performed in a chamber with an average of
25.degree. C. and 70% of air humidity.
Example 5
Microscopical Screening
[0512] For the evaluation of the pathogen development, the
inoculated leaves of plants were stained with aniline blue 48 hours
after infection.
[0513] The aniline blue staining serves for the detection of
fluorescent substances. During the defense reactions in host
interactions and non-host interactions, substances such as phenols,
callose or lignin accumulated or were produced and were
incorporated at the cell wall either locally in papillae or in the
whole cell (hypersensitive reaction, HR). Complexes were formed in
association with aniline blue, which lead e.g. in the case of
callose to yellow fluorescence. The leaf material was transferred
to falcon tubes or dishes containing destaining solution II
(ethanol/acetic acid 6/1) and was incubated in a water bath at
90.degree. C. for 10-15 minutes. The destaining solution II was
removed immediately thereafter, and the leaves were washed 2.times.
with water. For the staining, the leaves were incubated for 1.5-2
hours in staining solution 11 (0.05% aniline blue=methyl blue,
0.067 M di-potassium hydrogen phosphate) and analyzed by microscopy
immediately thereafter.
[0514] The different interaction types were evaluated (counted) by
microscopy. An Olympus UV microscope BX61 (incident light) and a UV
Longpath filter (excitation: 375/15, Beam splitter: 405 LP) are
used. After aniline blue staining, the spores appeared blue under
UV light. The papillae could be recognized beneath the fungal
appressorium by a green/yellow staining. The hypersensitive
reaction (HR) was characterized by a whole cell fluorescence.
Example 6
Evaluating the Susceptibility to Soybean Rust
[0515] The progression of the soybean rust disease was scored by
the estimation of the diseased area (area which was covered by
sporulating uredinia) on the backside (abaxial side) of the leaf.
Additionally the yellowing of the leaf was taken into account (for
scheme see FIG. 1).
[0516] At all 15 T0 soybean plants expressing MybTF protein were
inoculated with spores of Phakopsora pachyrhizi. The macroscopic
disease symptoms of soy against P. pachyrhizi of the inoculated
soybean plants were scored 14 days after inoculation.
[0517] The average of the percentage of the leaf area showing
fungal colonies or strong yellowing/browning on all leaves was
considered as diseased leaf area. At all 15 soybean To plants
expressing MybTF (expression checked by RT-PCR) were evaluated in
parallel to non-transgenic control plants. Non-transgenic soy
plants grown in parallel to the transgenic plants were used as
control. The average of the diseased leaf area is shown in FIG. 4
for plants expressing recombinant MybTF protein compared with
wildtype plants. Overexpression of the MybTF reduces the diseased
leaf area in comparison to non-transgenic control plants by 72.3%
in average over all events and plants generated. This data clearly
indicates that the in-planta expression of the MybTF expression
vector construct (see FIG. 2) lead to a lower disease scoring of
transgenic plants compared to non-transgenic controls. So, the
expression of MybTF nucleic acid (as shown in SEQ ID NO: 1) in
soybean significantly (p<0.001) increases the resistance of soy
against soybean rust.
Example 7
Construction of Maize Expression Cassettes
[0518] The nucleic acid sequence encoding the optimized cDNAs of
MYB-TF (as shown in SEQ ID NO:2 and SEQ ID NO:3) were synthesized
in a way that an Ascl restriction site is located in front of the
start-ATG and a Pstl restriction site downstream of the stop-codon.
The synthesized cDNAs were digested using the restriction enzymes
Ascl and Pstl (NEB Biolabs) and ligated in an Ascl/Pstl digested
binary plant transformation vector (description see below) in a way
that the full-length Myb-TF cDNA is located in sense direction
downstream of a SCBV254 promoter (Sugarcane Bacilliform Virus
promoter fragment ScBV-254) and upstream of a t-nos terminator
(3'UTR of Nopaline Synthase from Agrobacterium tumefaciens). An
intron from the rice Met1 gene was also cloned in between of the
promoter and the Myb-TF cDNA sequences.
[0519] As backbone a binary plant transformation vector was used,
which is composed of: (1) a Kanamycin resistance cassette for
bacterial selection (2) a pVS1 origin for replication in
Agrobacteria (3) a ColE1 origin of replication for stable
maintenance in E. coli and (4) between the right and left border a
ZmAHAS gene as selectable marker under control of a
ZmAHAS-promoter. A comprehensive description of the components of a
binary plant transformation vector can be found in the literature.
Examples for plant binary vectors are pBi-nAR (Hofgen &
Willmitzer 1990, Plant Sci. 66:221-230), pSUN300 or pSUN2-GW
vectors and the pPZP vectors (Hajdukiewicz et al., Plant Molecular
Biology 25: 989-994, 1994).
[0520] The recombinant binary plant transformation vectors
containing the Myb-TF cDNA expression cassettes were transformed
into Top10 cells (Invitrogen) using standard conditions.
Transformed cells were selected on LB agar containing 50 .mu.g/ml
kanamycin grown overnight at 37.degree. C. Plasmid DNA was
extracted using the QIAprep Spin Miniprep Kit (Qiagen) following
manufacturer's instructions. Analysis of subsequent clones and
restriction mapping was performed according to standard molecular
biology techniques (Sambrook et al., 1989, "Molecular Cloning: A
Laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).
Example 8
Maize Transformation
[0521] Agrobacterium cells harboring a plasmid containing the gene
of interest (see above) and the mutated maize AHAS gene were grown
in YP medium supplemented with appropriate antibiotics for 1-2
days. One loop of Agrobacterium cells was collected and suspended
in 1.8 ml M-LS-002 medium (LS-inf). The cultures were incubated
while shaking at 1,200 rpm for 5 min-3 hrs. Corn cobs were
harvested at 8-11 days after pollination. The cobs were sterilized
in 20% Clorox solution for 5 min, followed by spraying with 70%
Ethanol and then thoroughly rinsed with sterile water. Immature
embryos 0.8-2.0 mm in size were dissected into the tube containing
Agrobacterium cells in LS-inf solution.
[0522] The constructs were transformed into immature embryos by a
protocol modified from Japan Tobacco Agrobacterium mediated plant
transformation method (U.S. Pat. Nos. 5,591,616; 5,731,179;
6,653,529; and U.S. Patent Application Publication No.
2009/0249514). Two types of plasmid vectors were used for
transformation. One type had only one T-DNA border on each of left
and right side of the border, and selectable marker gene and gene
of interest were between the left and right T-DNA borders. The
other type was so called "two T-DNA constructs" as described in
Japan Tobacco U.S. Pat. No. 5,731,179. In the two DNA constructs,
the selectable marker gene was located between one set of T-DNA
borders and the gene of interest was included in between the second
set of T-DNA borders. Either plasmid vector can be used. The
plasmid vector was electroporated into Agrobacterium.
[0523] Agrobacterium infection of the embryos was carried out by
inverting the tube several times. The mixture was poured onto a
filter paper disk on the surface of a plate containing
cocultivation medium (M-LS-011). The liquid agro-solution was
removed and the embryos were checked under a microscope and placed
scutellum side up. Embryos were cultured in the dark at 22.degree.
C. for 2-4 days, and transferred to M-MS-101 medium without
selection and incubated for four to seven days. Embryos were then
transferred to M-LS-202 medium containing 0.75 .mu.M imazethapyr
and grown for three weeks at 27.degree. C. to select for
transformed callus cells.
[0524] Plant regeneration was initiated by transferring resistant
calli to M-LS-504 medium supplemented with 0.75 .mu.M imazethapyr
and growing under light at 26.degree. C. for two to three weeks.
Regenerated shoots were then transferred to a rooting box with
M-MS-618 medium (0.5 .mu.M imazethapyr). Plantlets with roots were
transferred to soil-less potting mixture and grown in a growth
chamber for a week, then transplanted to larger pots and maintained
in a greenhouse until maturity.
[0525] Transgenic maize plant production is also described, for
example, in U.S. Pat. Nos. 5,591,616 and 6,653,529; U.S. Patent
Application Publication No. 2009/0249514; and WO/2006136596, each
of which are hereby incorporated by reference in their entirety.
Transformation of maize may be made using Agrobacterium
transformation, as described in U.S. Pat. Nos. 5,591,616;
5,731,179; U.S. Patent Application Publication No. 2002/0104132,
and the like. Transformation of maize (Zea mays L.) can also be
performed with a modification of the method described by Ishida et
al. (Nature Biotech., 1996, 14:745-750). The inbred line A188
(University of Minnesota) or hybrids with A188 as a parent are good
sources of donor material for transformation (Fromm et al.,
Biotech, 1990, 8:833), but other genotypes can be used successfully
as well. Ears are harvested from corn plants at approximately 11
days after pollination (DAP) when the length of immature embryos is
about 1 to 1.2 mm. Immature embryos are co-cultivated with
Agrobacterium tumefaciens that carry "super binary" vectors and
transgenic plants are recovered through organogenesis. The super
binary vector system is described in WO 94/00977 and WO 95/06722.
Vectors are constructed as described. Various selection marker
genes are used including the maize gene encoding a mutated
acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541).
Similarly, various promoters are used to regulate the trait gene to
provide constitutive, developmental, inducible, tissue or
environmental regulation of gene transcription. Excised embryos can
be used and can be grown on callus induction medium, then maize
regeneration medium, containing imidazolinone as a selection agent.
The Petri dishes are incubated in the light at 25.degree. C. for
2-3 weeks, or until shoots develop. The green shoots are
transferred from each embryo to maize rooting medium and incubated
at 25.degree. C. for 2-3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are
produced from plants that exhibit tolerance to the imidazolinone
herbicides and which are PCR positive for the transgenes.
Example 9
Fusarium and Colletotrichum Resistance Screening
[0526] Transgenic maize plants expressing the Myb-TF cDNAs (SEQ ID
NO:2 and SEQ ID NO:3), under control of the constitutive SCBV254
promoter (Sugarcane Bacilliform Virus promoter fragment ScBV-254),
were grown in greenhouse or phyto-chamber under standard growing
conditions in a controlled environment (20-25.degree. C., 60-90%
humidity).
[0527] Shortly after the transgenic maize plants enter the
reproductive phase they are inoculated near the base of the stalk
using a fungal suspension of spores (10.sup.5 spores in PBS
solution) of Fusarium ssp. or Colletotrichum graminicola. Plants
are incubated for 2-4 weeks at 20-25.degree. C. and 60-90%
humidity.
[0528] For scoring the stalk rot disease, stalks are split and the
progression of the disease is scored by observation of the
characteristic brown to black color of the fungus as it grows up
the stalk. Disease ratings are conducted by assigning a visual
score. Per experiment the diseased leaf area of more than 10
transgenic plants (and wild-type plants as control) is scored. For
analysis the average of the diseased leaf area of the
non-transgenic mother plant is set to 100% to calculate the
relative diseased leaf area of the transgenic lines
[0529] The expression of the Myb-TF gene will lead to enhanced
resistance of corn against Fusarium ssp. and Colletotrichum
graminicola.
Sequence CWU 1
1
5611187DNAArabidopsis thalianamisc_feature(1)..(1187)Nucleotide
sequence of the MybTF gene; mol_type = unassigned DNA 1atgaagatgg
atttttcatg tttccaagaa tacccttttg agtttcattg cagaggaaca 60acatttaatg
ggtttagaga aaacaatgca gtgtctgaaa cagtagaaga gttctgtaat
120aaaagaagga tgcagaagaa gagtgatgat ttgaaaacta agaagaagaa
gaaacagagt 180gtttctaggg tttgtagtag aggacattgg aggatctctg
aagatactca gcttatggag 240cttgtttcgg tttacggtcc tcaaaactgg
aaccacattg cagagagtat gcaaggaaga 300acaggtaacg acaaaaattg
aaatctttaa tcttccctta gctaattccg gaacatgaaa 360cttacaatgt
tttttcttgc ttctttgtgt tttgtcttaa aggaaagagc tgcagattga
420ggtggtttaa ccagttagat ccgaggatta acaagagagc tttcagtgat
gaagaagaag 480agagactact tgctgctcat agagcttttg gtaacaaatg
ggctatgatt gctaagcttt 540tcaatggaag aacagataat gccttgaaga
atcattggca tgttctcatg gcaaggaaga 600tgagacagca atcaagttct
tacgtccaaa gattcaatgg ttctgctcat gaatctaaca 660cagatcacaa
aatcttcaat ctttctcctg gtttgtctct tcttacctta cacatatgca
720ttgagtttaa ctctgttatt gtaatgagat actttcgata tttatcactc
aggaacaatg 780aacttatggt ttggtcacaa aagtagtcag attgcaagtt
tggtgagtct ttaagtttca 840tggttctgtg tgttcttgca ggtaatgtag
atgatgatga agatgtgaat ctgaaaaagt 900gcagctggga aatgctaaaa
gagggaacta ctaacctgaa agctcagtat ctccaagaag 960aatatagttc
ttcacgcatg ccgatgcagg gtccacatca tcactactca accttccctg
1020cagattcctt ggcactgaca ctgcatgtct ccatccagga accatcatca
tcatcgtcat 1080tatcactgcc atcatcatca acaactggag aacatacaat
ggtgaccaga tattttgaaa 1140ccattaaacc tccagcattt atagattttc
taggagttgg tcactaa 11872708DNAArtificial SequenceSynthetic
polynucleotide 2atgaagatgg actttagctg ctttcaagag taccccttcg
agtttcactg taggggcact 60acctttaacg gctttagaga gaacaacgcc gttagcgaga
ctgtggaaga gttctgtaac 120aagcgtagga tgcagaagaa gtcagacgac
cttaagacta agaagaagaa gaaacagtca 180gttagtaggg tgtgctctag
gggccattgg aggattagtg aggatactca gctcatggaa 240ctcgtttcag
tttacggacc tcagaactgg aatcatattg ccgagtctat gcaaggtagg
300accggtaaga gttgtaggct tcgttggttt aatcagctcg accctaggat
taacaagagg 360gcctttagtg acgaagagga agagaggctt cttgctgctc
acagggcttt cggtaacaag 420tgggctatga tcgctaagct ctttaacggt
aggaccgata acgcccttaa gaatcactgg 480cacgtgctca tggctaggaa
gatgaggcaa cagagttcta gctacgttca gaggtttaac 540ggctcagctc
acgagtctaa caccgatcac aagatcttta accttagccc aggccttagc
600ctcctaaccc ttcatatctg tatcgagttt aactcagtga tcgtgatgag
atactttaga 660taccttagcc ttaggaacaa cgagctgatg gtgtggtcac agaagtga
7083918DNAArtificial SequenceSynthetic polynucleotide 3atgaagatgg
actttagctg ctttcaagag taccccttcg agtttcactg taggggcact 60acctttaacg
gctttagaga gaacaacgcc gttagcgaga ctgtggaaga gttctgtaac
120aagcgtagga tgcagaagaa gtcagacgac cttaagacta agaagaagaa
gaaacagtca 180gttagtaggg tgtgctctag gggccactgg cgtattagtg
aggatactca gctaatggaa 240ctagtttcag tctacggccc tcagaactgg
aatcatatag ccgagtctat gcagggtagg 300accggtaagt cttgtaggct
tcgttggttt aatcagctag accctaggat taacaagcgc 360gcctttagtg
acgaagagga agagagacta ctagccgctc acagggcttt cggtaacaag
420tgggctatga tagctaagct ctttaacggt aggaccgata acgcccttaa
gaatcactgg 480cacgtgctaa tggctaggaa gatgaggcag cagagttcta
gctacgttca gcgctttaac 540ggatcagctc acgagtctaa caccgatcac
aagatcttta accttagccc cggtaacgtg 600gacgacgacg aggacgttaa
ccttaaaaag tgcagttggg agatgcttaa agagggcact 660actaacctta
aggctcagta ccttcaagaa gagtactcta gctctaggat gcctatgcag
720ggacctcacc accactactc taccttccca gctgatagcc tagctctaac
ccttcacgtt 780agtattcaag agcctagcag ttctagtagc cttagcctac
ctagcagttc aactaccggt 840gagcacacta tggtcactag atacttcgag
actattaagc ccccagcctt tatagacttt 900ctaggcgttg gtcactaa
9184918DNAArabidopsis thalianamisc_feature(1)..(918)Nucleotide
sequence of the second CDS (CDS2) sequence (At3g29020.2) of the
MybTF gene; mol_type = unassigned DNA 4atgaagatgg atttttcatg
tttccaagaa tacccttttg agtttcattg cagaggaaca 60acatttaatg ggtttagaga
aaacaatgca gtgtctgaaa cagtagaaga gttctgtaat 120aaaagaagga
tgcagaagaa gagtgatgat ttgaagacta agaagaagaa gaaacagagt
180gtttctaggg tttgtagtag aggacattgg aggatctctg aagatactca
gcttatggag 240cttgtttcgg tttacggtcc tcaaaactgg aaccacattg
cagagagtat gcaaggaaga 300acaggaaaga gctgcagatt gaggtggttt
aaccagttag atccgaggat taacaagaga 360gctttcagtg atgaagaaga
agagagacta cttgctgctc atagagcttt tggtaacaaa 420tgggctatga
ttgctaagct tttcaatgga agaacagata atgccttgaa gaatcattgg
480catgttctca tggcaaggaa gatgagacag caatcaagtt cttacgtcca
aagattcaat 540ggttctgctc atgaatctaa cacagatcac aaaatcttca
atctttctcc tggtaatgta 600gatgatgatg aagatgtgaa tctgaaaaag
tgcagctggg aaatgctaaa agagggaact 660actaacctga aagctcagta
tctccaagaa gaatatagtt cttcacgcat gccgatgcag 720ggtccacatc
atcactactc aaccttccct gcagattcct tggcactgac actgcatgtc
780tccatccagg aaccatcatc atcatcgtca ttatcactgc catcatcatc
aacaactgga 840gaacatacaa tggtgaccag atattttgag accattaaac
ctccagcatt tatagatttt 900ctaggagttg gtcactaa 9185305PRTArabidopsis
thalianamisc_featureAmino acid sequence of the MybTF protein as
derived from CDS2 nucleotide sequence 5Met Lys Met Asp Phe Ser Cys
Phe Gln Glu Tyr Pro Phe Glu Phe His 1 5 10 15 Cys Arg Gly Thr Thr
Phe Asn Gly Phe Arg Glu Asn Asn Ala Val Ser 20 25 30 Glu Thr Val
Glu Glu Phe Cys Asn Lys Arg Arg Met Gln Lys Lys Ser 35 40 45 Asp
Asp Leu Lys Thr Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val 50 55
60 Cys Ser Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu
65 70 75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala
Glu Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg
Trp Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile Asn Lys Arg Ala Phe
Ser Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu Ala Ala His Arg Ala
Phe Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala Lys Leu Phe Asn Gly
Arg Thr Asp Asn Ala Leu Lys Asn His Trp 145 150 155 160 His Val Leu
Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val 165 170 175 Gln
Arg Phe Asn Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile 180 185
190 Phe Asn Leu Ser Pro Gly Asn Val Asp Asp Asp Glu Asp Val Asn Leu
195 200 205 Lys Lys Cys Ser Trp Glu Met Leu Lys Glu Gly Thr Thr Asn
Leu Lys 210 215 220 Ala Gln Tyr Leu Gln Glu Glu Tyr Ser Ser Ser Arg
Met Pro Met Gln 225 230 235 240 Gly Pro His His His Tyr Ser Thr Phe
Pro Ala Asp Ser Leu Ala Leu 245 250 255 Thr Leu His Val Ser Ile Gln
Glu Pro Ser Ser Ser Ser Ser Leu Ser 260 265 270 Leu Pro Ser Ser Ser
Thr Thr Gly Glu His Thr Met Val Thr Arg Tyr 275 280 285 Phe Glu Thr
Ile Lys Pro Pro Ala Phe Ile Asp Phe Leu Gly Val Gly 290 295 300 His
305 6702DNAArabidopsis thalianasource(1)..(702)Nucleotide sequence
of the first CDS (CDS1) sequence (At3g29020.1) of the MybTF gene;
mol_type = unassigned DNA 6atggattttt catgtttcca agaataccct
tttgagtttc attgcagagg aacaacattt 60aatgggttta gagaaaacaa tgcagtgtct
gaaacagtag aagagttctg taataaaaga 120aggatgcaga agaagagtga
tgatttgaag actaagaaga agaagaaaca gagtgtttct 180agggtttgta
gtagaggaca ttggaggatc tctgaagata ctcagcttat ggagcttgtt
240tcggtttacg gtcctcaaaa ctggaaccac attgcagaga gtatgcaagg
aagaacagga 300aagagctgca gattgaggtg gtttaaccag ttagatccga
ggattaacaa gagagctttc 360agtgatgaag aagaagagag actacttgct
gctcatagag cttttggtaa caaatgggct 420atgattgcta agcttttcaa
tggaagaaca gataatgcct tgaagaatca ttggcatgtt 480ctcatggcaa
ggaagatgag acagcaatca agttcttacg tccaaagatt caatggttct
540gctcatgaat ctaacacaga tcacaaaatc ttcaatcttt ctcctggttt
gtctcttctt 600accttacaca tatgcattga gtttaactct gttattgtaa
tgagatactt tcgatattta 660tcactcagga acaatgaact tatggtttgg
tctcaaaagt ag 7027233PRTArabidopsis thalianamisc_featureAmino acid
sequence of the MybTF protein as derived from CDS1 nucleotide
sequence 7Met Asp Phe Ser Cys Phe Gln Glu Tyr Pro Phe Glu Phe His
Cys Arg 1 5 10 15 Gly Thr Thr Phe Asn Gly Phe Arg Glu Asn Asn Ala
Val Ser Glu Thr 20 25 30 Val Glu Glu Phe Cys Asn Lys Arg Arg Met
Gln Lys Lys Ser Asp Asp 35 40 45 Leu Lys Thr Lys Lys Lys Lys Lys
Gln Ser Val Ser Arg Val Cys Ser 50 55 60 Arg Gly His Trp Arg Ile
Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65 70 75 80 Ser Val Tyr Gly
Pro Gln Asn Trp Asn His Ile Ala Glu Ser Met Gln 85 90 95 Gly Arg
Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe Asn Gln Leu Asp 100 105 110
Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu Arg Leu 115
120 125 Leu Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile Ala
Lys 130 135 140 Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His
Trp His Val 145 150 155 160 Leu Met Ala Arg Lys Met Arg Gln Gln Ser
Ser Ser Tyr Val Gln Arg 165 170 175 Phe Asn Gly Ser Ala His Glu Ser
Asn Thr Asp His Lys Ile Phe Asn 180 185 190 Leu Ser Pro Gly Leu Ser
Leu Leu Thr Leu His Ile Cys Ile Glu Phe 195 200 205 Asn Ser Val Ile
Val Met Arg Tyr Phe Arg Tyr Leu Ser Leu Arg Asn 210 215 220 Asn Glu
Leu Met Val Trp Ser Gln Lys 225 230 81340DNAArabidopsis
thalianamisc_feature(1)..(1340)Nucleotide sequence of the
full-length genomic MybTF sequence (TAIR accession No 4010724011);
mol_type = unassigned DNA 8gtatatatga gacattagtt atagaagaga
gactaacatg aagatggatt tttcatgttt 60ccaagaatac ccttttgagt ttcattgcag
aggaacaaca tttaatgggt ttagagaaaa 120caatgcagtg tctgaaacag
tagaagagtt ctgtaataaa agaaggatgc agaagaagag 180tgatgatttg
aagactaaga agaagaagaa acagagtgtt tctagggttt gtagtagagg
240acattggagg atctctgaag atactcagct tatggagctt gtttcggttt
acggtcctca 300aaactggaac cacattgcag agagtatgca aggaagaaca
ggtaacgaca aaaattgaaa 360tctttaatct tcccttagct aattccggaa
catgaaactt acaatgtttt ttcttgcttc 420tttgtgtttt gtcttaaagg
aaagagctgc agattgaggt ggtttaacca gttagatccg 480aggattaaca
agagagcttt cagtgatgaa gaagaagaga gactacttgc tgctcataga
540gcttttggta acaaatgggc tatgattgct aagcttttca atggaagaac
agataatgcc 600ttgaagaatc attggcatgt tctcatggca aggaagatga
gacagcaatc aagttcttac 660gtccaaagat tcaatggttc tgctcatgaa
tctaacacag atcacaaaat cttcaatctt 720tctcctggtt tgtctcttct
taccttacac atatgcattg agtttaactc tgttattgta 780atgagatact
ttcgatattt atcactcagg aacaatgaac ttatggtttg gtctcaaaag
840tagtcagatt gcaagtttgg tgagtcttta agtttcatgg ttctgtgtgt
tcttgcaggt 900aatgtagatg atgatgaaga tgtgaatctg aaaaagtgca
gctgggaaat gctaaaagag 960ggaactacta acctgaaagc tcagtatctc
caagaagaat atagttcttc acgcatgccg 1020atgcagggtc cacatcatca
ctactcaacc ttccctgcag attccttggc actgacactg 1080catgtctcca
tccaggaacc atcatcatca tcgtcattat cactgccatc atcatcaaca
1140actggagaac atacaatggt gaccagatat tttgagacca ttaaacctcc
agcatttata 1200gattttctag gagttggtca ctaaagctct aacatttaga
gtgggaacta atcaagaagt 1260tgcttactcc tgtcattatt atcaaagtct
ctgacttttc ttttgttagc cattaacatg 1320acaagctaaa gacatcaagt
13409708DNAArtificial SequenceSynthetic polynucleotide 9atgaaaatgg
atttcagttg tttccaggaa tatccttttg aattccattg ccgagggacg 60acattcaatg
gattccggga aaataatgcg gtatcggaaa cggtggagga attttgcaat
120aaaagacgaa tgcaaaaaaa atctgatgat ctaaaaacga aaaaaaaaaa
gaagcaaagt 180gtcagccgcg tttgttcccg gggacactgg cgcatatctg
aagacacgca attgatggag 240ctagtctctg tctatggacc acaaaattgg
aaccacatcg cggaatccat gcagggacga 300acagggaaat cctgccggct
cagatggttc aaccaattgg atccacgtat caacaaacgt 360gcgttctctg
atgaggaaga ggaacgtctc ttagcagcgc atcgggcctt tgggaataaa
420tgggcaatga ttgccaaatt attcaatggg cgtactgaca atgcgttgaa
aaaccactgg 480catgtgttga tggcgcgtaa aatgagacag caatcctcgt
cttatgtcca acggttcaat 540ggatccgcac atgaatcgaa tacggaccat
aaaatattca atttatctcc tgggttgtcg 600cttttgacgc tacacatttg
catagaattc aattcggtta ttgtcatgag atatttcagg 660tatctgtctt
tacgcaataa tgaaatgatg gtatggtctc aaaaatag 70810708DNAArtificial
SequenceSynthetic polynucleotide 10atgaaaatgg atttctcctg tttccaggaa
tatccttttg agttccattg caggggaacc 60actttcaatg ggttccgaga aaataacgct
gtctcagaaa cggtcgaaga attttgtaat 120aaacgcagaa tgcaaaagaa
atctgacgat ctcaaaacaa aaaagaagaa aaaacaatcc 180gtatcccgag
tatgctccag agggcattgg cgaataagtg aagatactca gttgatggag
240ttagtgagcg tatatggacc tcagaattgg aaccacatcg cggaatcaat
gcaggggaga 300acgggaaaaa gctgccggct acgctggttc aatcagttgg
atcctcgtat taataaaagg 360gccttcagcg atgaggaaga ggaacgactc
ctggctgcgc atcgggcatt cggaaataag 420tgggcaatga ttgccaagtt
gttcaacggt cggacggaca atgcccttaa aaaccattgg 480cacgttctaa
tggcgaggaa aatgcggcag caaagttcca gttatgtcca acgtttcaat
540ggctccgcgc atgaatccaa tacagaccat aaaatattca atctatctcc
aggcctgtct 600ctattaacac tacacatatg cattgaattc aactccgtca
tagttatgcg ttattttagg 660tatttgtctt tacgcaataa tgaaatgatg
gtatggagcc agaaatga 70811708DNAArtificial SequenceSynthetic
polynucleotide 11atgaagatgg atttcagttg tttccaggag tatccctttg
aatttcattg tagagggact 60acctttaacg ggttccgcga aaacaatgcc gtttccgaaa
ccgtggagga gttttgtaat 120aaacgtagaa tgcaaaaaaa aagcgacgat
cttaagacca aaaaaaaaaa gaagcagtcc 180gtgagccgcg tgtgttcaag
gggccactgg cgtatcagtg aggacactca attaatggag 240ctcgtatccg
tatacggccc gcaaaattgg aatcatattg cagagtccat gcaggggaga
300accggaaagt catgccgtct taggtggttc aaccaactag atcctcgaat
aaataaacgg 360gccttcagtg atgaggaaga ggaacggcta cttgctgccc
acagggcttt tggaaataaa 420tgggccatga ttgcaaagct cttcaatggc
aggaccgaca atgcgttaaa aaaccattgg 480catgtgctca tggctcggaa
aatgagacaa caaagtagta gctatgttca gcgcttcaat 540ggcagtgctc
atgaatcaaa cacggaccac aaaattttca atttgtctcc tggcctctct
600ttactaacct tacacatttg tatcgaattc aatagcgtga tcgtcatgcg
gtacttccgc 660taccttagcc tcaggaacaa cgagctgatg gtttggtcac aaaaatga
70812708DNAArtificial SequenceSynthetic polynucleotide 12atgaaaatgg
atttctcatg ctttcaggag taccccttcg aattccactg cagaggcacc 60acattcaatg
ggttcagaga gaacaacgcc gtctcggaga ctgtagaaga gttctgtaat
120aaacggcgca tgcagaaaaa atcagacgat cttaagacca aaaagaagaa
aaaacaatca 180gttagtaggg tttgcagcag ggggcactgg cggattagtg
aggatacgca gctgatggag 240ctcgtctcgg tatatggtcc acaaaactgg
aaccatattg cggagtctat gcaggggcgg 300acaggcaaga gttgtaggct
tcgttggttt aatcaactcg atcctcgcat aaacaagcgg 360gctttttcgg
atgaagaaga ggagaggctt ctagctgctc accgggcctt cggtaataaa
420tgggcgatga tcgcgaagct cttcaatggt cgtaccgaca atgccctgaa
gaatcattgg 480catgtgctca tggctcgtaa gatgaggcag cagagttcta
gctacgtaca gcgcttcaat 540gggtcggctc acgagtccaa caccgatcat
aaaatcttta accttagccc cgggctgagc 600ttattaacac tccatatttg
tattgaattc aatagtgtga tcgtgatgag gtactttaga 660tacctgagtt
tacggaataa cgagatgatg gtgtggtcac agaagtga 70813708DNAArtificial
SequenceSynthetic polynucleotide 13atgaagatgg actttagctg cttccaagaa
taccccttcg aatttcactg caggggtact 60acgttcaacg gctttagaga aaacaacgca
gttagcgaaa ctgtggagga gttctgcaac 120aaacgtagga tgcagaagaa
gtcagatgac ttgaaaacta aaaagaaaaa gaagcaaagc 180gttagtaggg
tgtgttctag ggggcattgg aggatttccg aagatactca gctgatggag
240ctcgttagcg tttacggccc tcagaactgg aaccacattg ccgagtctat
gcagggtagg 300accggaaagt cctgtaggct tcgttggttt aatcagctcg
accctaggat taacaagcgg 360gcttttagtg acgaggagga ggagaggctt
ctagctgctc atcgggcttt cggtaacaag 420tgggctatga tcgctaaatt
attcaacggt cgtaccgata acgcccttaa gaaccattgg 480cacgtgctca
tggcacgcaa gatgaggcag cagagttcta gctatgttca gaggtttaac
540ggctcagctc acgagtctaa cacagaccat aagattttta atctgtcacc
aggccttagt 600ctactaacct tacatatatg tattgagttt aatagcgtga
tagtgatgag atattttaga 660taccttagcc taaggaacaa cgaactgatg
gtctggtcac aaaagtga 70814708DNAArtificial SequenceSynthetic
polynucleotide 14atgaagatgg acttttcctg ctttcaagag taccccttcg
agtttcactg taggggcact 60acctttaacg gcttcagaga gaataacgca gttagcgaga
ctgtagagga gttctgtaat 120aagcgtcgta tgcagaagaa gtcagatgac
ctcaagacta agaagaagaa aaaacaaagt 180gtgagtaggg tgtgctctag
gggccattgg aggatcagtg aggatacgca gctcatggag 240cttgtttcag
tttacggacc tcaaaactgg aaccatatcg ccgaatctat gcaaggtagg
300accggtaaga gttgtaggct tcgttggttc aatcagctag acccgcgaat
caacaagagg 360gcctttagtg acgaagagga agagaggctt ttagctgctc
acagggcatt tggtaataag 420tgggctatga tcgccaagtt gtttaacggt
aggacggaca acgcgcttaa gaatcactgg 480cacgtgctca tggcaaggaa
gatgaggcaa caaagttctt catatgtcca gcgttttaat 540ggcagtgctc
acgaatctaa caccgatcac aagatcttca accttagccc cgggcttagc
600ctcctaaccc ttcacatctg tatcgagttc aacagcgtca tcgtgatgag
atattttcgc 660taccttagcc ttaggaataa cgagctgatg gtgtggtcac
aaaaatga
70815708DNAArtificial SequenceSynthetic polynucleotide 15atgaagatgg
attttagctg tttccaagag taccccttcg agtttcactg caggggcact 60accttcaacg
gctttagaga gaacaacgcc gttagcgaga ctgtggaaga gttctgtaac
120aagcgtagga tgcagaaaaa gtcggacgac ctcaagacta aaaagaagaa
gaaacagtca 180gttagtaggg tgtgctcgag aggccattgg aggattagtg
aggacacgca gctcatggaa 240ctcgtttcag tgtacggacc tcagaattgg
aaccatattg cagagtctat gcaaggtagg 300actggtaaaa gttgcaggct
tcgttggttt aaccagctcg atcctcgtat taacaagagg 360gcctttagtg
acgaagagga agagaggctt cttgctgctc acagggcttt cgggaataag
420tgggctatga tcgctaagct ctttaacggt aggaccgata acgctcttaa
gaaccactgg 480cacgtactca tggctaggaa gatgaggcaa cagagttcta
gctacgttca gaggtttaat 540ggctcggctc acgaatctaa cacggatcac
aagatcttta acttgagccc aggccttagc 600ctcctaaccc tgcatatctg
tatcgagttt aactcagtga tcgtgatgag atactttaga 660taccttagcc
ttaggaacaa cgaactgatg gtgtggtcgc agaagtga 70816708DNAArtificial
SequenceSynthetic polynucleotide 16atgaagatgg acttcagctg ctttcaagag
taccccttcg agtttcactg taggggcact 60acctttaacg gcttcagaga gaacaacgcc
gttagcgaaa ctgtggaaga gttctgtaac 120aagcgtagga tgcagaagaa
atcagacgac cttaagacta agaagaagaa gaagcagtca 180gttagtaggg
tgtgctctag gggccattgg aggattagtg aggatactca gctgatggaa
240ctcgtttcag tgtacggacc tcagaactgg aatcacattg ccgagtctat
gcaaggtagg 300accggtaagt cgtgtaggct tcgttggttt aatcagctcg
accctcggat taacaagagg 360gcctttagtg acgaagagga agagaggctt
ctagcagctc acagggcttt cggtaacaag 420tgggctatga tcgctaagct
ctttaatggt aggaccgata atgcccttaa aaatcactgg 480catgtgctca
tggctaggaa gatgaggcaa cagagttcta gctacgttca gcgctttaac
540ggctcagctc acgagtctaa caccgatcac aagatcttta acctttctcc
cggccttagc 600ctcctaaccc ttcatatctg tatcgagttt aattcagtga
ttgtgatgag atactttaga 660tacctaagcc ttaggaacaa cgagctgatg
gtgtggtcac agaagtga 70817918DNAArtificial SequenceSynthetic
polynucleotide 17atgaaaatgg atttctcctg tttccaggaa tatccatttg
aattccattg cagagggacc 60actttcaatg gcttcagaga aaataatgca gtatcagaaa
ccgttgagga attttgcaat 120aaacgaagaa tgcaaaaaaa atccgatgat
ctgaaaacca aaaaaaaaaa aaagcaatca 180gtgagccgcg tctgttctcg
aggacattgg agaataagcg aagacacaca actcatggag 240ctggtaagcg
tatatggacc gcaaaattgg aaccatattg ctgaatccat gcaaggcaga
300acaggcaaaa gttgccgtct gagatggttc aatcaattgg atccacgcat
aaataaaagg 360gcgttcagcg atgaggaaga ggaaaggctt ctcgctgccc
acagagcgtt tggcaataaa 420tgggcgatga ttgccaaatt gttcaatggc
agaacggaca atgcattgaa aaaccattgg 480catgtcttaa tggcccgtaa
aatgaggcaa caatcctcat cttatgtcca acgtttcaat 540ggcagcgcac
atgaatccaa tacagaccat aaaatcttca atctatcgcc tggcaatgtt
600gatgatgatg aagatgtaaa tctaaagaaa tgttcctggg aaatgctgaa
ggaaggtaca 660acgaatctaa aggcccaata tctccaggag gaatatagtt
ccagtcgaat gccaatgcaa 720ggcccgcatc atcattattc cacttttccc
gcggatagtt tggctcttac attacatgta 780tccatacagg aaccctcttc
gagctcctcc ctgtcattac cgtcttcaag caccacaggg 840gaacatacga
tggttacgcg ttattttgaa acaataaaac cgcccgcgtt cattgatttc
900ttaggtgtgg ggcattag 91818918DNAArtificial SequenceSynthetic
polynucleotide 18atgaaaatgg acttcagttg tttccaagaa tatcctttcg
aattccattg caggggcacc 60acgtttaatg gcttcagaga gaataatgcg gtaagtgaaa
ccgtcgagga attttgcaat 120aaaagaagaa tgcaaaaaaa gagcgacgat
ttaaaaacaa aaaagaaaaa aaagcaaagc 180gtcagtcgtg tctgtagtcg
cggacattgg cgtatctccg aagatacgca actcatggag 240ctcgtctccg
tgtatggtcc gcaaaattgg aaccatattg ccgaaagcat gcaaggacgt
300accgggaaat cttgcagatt gcggtggttc aaccagctcg atcctagaat
aaataaacga 360gccttctctg acgaggaaga ggaacgccta cttgcagcgc
atagagcgtt cggaaataaa 420tgggcgatga ttgcgaagct ttttaatggg
aggaccgata acgccttaaa aaatcattgg 480catgtcttaa tggcgaggaa
aatgcgtcaa caatcatcta gctatgtcca acggtttaat 540ggttcggcgc
atgaatcgaa tacagaccat aaaatattca atttgtcccc tggcaacgta
600gatgatgatg aggatgttaa tctcaagaaa tgcagttggg agatgctaaa
ggaagggacg 660accaatttaa aagctcaata tttgcaagag gagtatagta
gctcacgcat gcccatgcaa 720ggcccccacc accattattc gacgtttcca
gcggatagtt tagcccttac attgcatgta 780tccatacagg agcccagtag
ctcctctagt ctttcgctgc cctcctcttc aaccacggga 840gaacatacca
tggttacgcg ttattttgaa acgatcaagc ccccggcttt cattgatttc
900ctgggagtcg gtcattaa 91819918DNAArtificial SequenceSynthetic
polynucleotide 19atgaaaatgg atttcagctg tttccaggaa tacccctttg
aatttcactg taggggcact 60acgtttaatg gcttcagaga aaataatgct gtttcggaga
ccgtagaaga attttgcaat 120aaacgccgga tgcaaaagaa aagtgatgac
ttgaaaacga agaagaaaaa aaagcaatca 180gtttcccgcg tgtgttcaag
ggggcactgg cgaatcagtg aagacactca gctaatggag 240ttagtttcag
tctatggtcc gcaaaactgg aaccatatag ctgagtcgat gcaaggtcgt
300acagggaagt catgtcgact tagatggttc aaccagttgg atcctcgcat
caacaagcgt 360gcgtttagtg atgaggagga ggaacgtcta ctagccgccc
atcgagcgtt tggaaacaag 420tgggctatga tcgcgaagtt attcaatgga
cggactgaca acgcccttaa aaaccattgg 480catgtattaa tggcaaggaa
aatgcgtcaa cagtcttcta gctacgttca gagattcaac 540ggatcagccc
acgaatcaaa tacagaccat aaaattttta acctatcgcc tgggaatgta
600gatgatgacg aagatgttaa cctcaaaaaa tgttcatggg agatgctaaa
agaagggacg 660acaaatttaa aagcccagta tttacaagaa gaatatagtt
cctctcgtat gccaatgcaa 720ggccctcacc accactacag tacatttcca
gcagactcct tggctttgac tctacacgtg 780tccatacagg aaccgtcctc
ttcttcaagc cttagcctac ctagcagttc aaccaccggg 840gaacatacca
tggtgacaag atattttgaa accataaaac cacctgcatt catagatttc
900ctaggcgtgg gtcactaa 91820918DNAArtificial SequenceSynthetic
polynucleotide 20atgaaaatgg actttagctg ctttcaagag tacccatttg
aatttcactg tcggggcact 60acgtttaacg gcttcagaga gaataacgcg gtttccgaga
ctgtggagga gttctgcaac 120aaacgtagga tgcaaaagaa gtcggacgac
ctaaagacga aaaagaagaa aaaacaatca 180gtcagtaggg tctgtagtcg
aggccactgg aggattagtg aagacacaca gctaatggag 240ctggtatcag
tctacgggcc tcaaaattgg aaccacatag ccgagagtat gcaaggtagg
300actggtaagt cttgtaggct taggtggttc aaccaacttg acccgcggat
taacaagcgc 360gccttcagcg atgaagagga agaaagactg ttggctgctc
atagggcgtt cggcaacaag 420tgggccatga tagctaagct cttcaatgga
aggaccgaca acgccttaaa gaatcactgg 480catgtgttaa tggcacgtaa
gatgaggcaa caaagttctt cgtacgtgca gaggttcaac 540ggtagtgcac
acgagtcgaa caccgaccac aagatattca atcttagtcc cggaaatgtg
600gatgatgacg aggatgttaa cctaaagaaa tgctcatggg aaatgcttaa
ggagggtaca 660actaatctta aggctcagta ccttcaggag gaatactcca
gttctcgcat gcccatgcaa 720ggtcctcacc atcactactc tacgtttcca
gcagattcgt tagccttgac gctacatgtt 780agtatacaag aaccttcatc
ttctagtagt ctttctctcc ctagcagttc aacgacgggt 840gagcatacga
tggtcactcg gtatttcgag acgataaaac cgccagcctt catagacttc
900ttaggggtag gtcactaa 91821918DNAArtificial SequenceSynthetic
polynucleotide 21atgaagatgg actttagctg ctttcaagag tatccattcg
agtttcactg ccgggggacg 60actttcaacg ggtttagaga aaataacgcc gttagcgaga
ctgtagaaga gttctgtaat 120aagcgtagga tgcagaagaa gtcggacgac
ctgaaaacga aaaagaagaa gaaacagtca 180gtttcgcggg tgtgttcaag
ggggcactgg cggatcagtg aggatactca gttaatggag 240ttagtttcag
tctacggccc tcagaactgg aatcatattg ccgagtcaat gcagggtcgc
300accggcaagt cttgtaggct tcgctggttt aaccagttgg accctaggat
taataagcgc 360gccttcagtg acgaagagga agagagactc ttagccgcgc
acagggcttt cggtaacaag 420tgggcaatga ttgctaaact ctttaacggg
aggactgata atgcccttaa gaatcattgg 480catgtgctaa tggctagaaa
gatgaggcaa cagagttcta gctatgtaca acggttcaac 540ggttcagcgc
acgagtccaa cacagatcat aaaatcttca acctttcgcc cggcaatgtg
600gacgacgacg aggatgttaa cctgaagaag tgctcctggg agatgctgaa
agagggcact 660acgaacctta aggctcagta tctccaagaa gagtactcta
gctctaggat gcctatgcaa 720ggacctcacc accactattc taccttccca
gccgatagcc tagctctaac cctacacgtc 780tccattcaag agccttccag
ttcttcaagc ctttccctac ctagcagtag cactaccggt 840gaacacacta
tggtcacgag atacttcgaa accattaagc caccagcctt tatagacttc
900cttggcgtag gtcactga 91822918DNAArtificial SequenceSynthetic
polynucleotide 22atgaagatgg attttagctg ctttcaagag tacccctttg
aattccactg tcgtggcact 60acctttaacg ggtttagaga gaacaacgcc gtgtctgaga
ctgtggaaga gttctgtaac 120aagcgtagga tgcagaagaa gtcagatgac
cttaaaacta agaaaaagaa gaagcagtca 180gttagtcgtg tgtgcagtag
gggccactgg agaattagtg aggatactca actaatggag 240ctagtctcag
tctacggccc tcaaaactgg aatcacatag ccgagtccat gcagggacgt
300accggtaagt cttgcaggct tcgttggttc aatcaattgg accctaggat
taataagcgc 360gcctttagtg acgaagagga agagagactt ctagcagctc
atcgagcttt cggtaataag 420tgggctatga tagctaagct ctttaatggt
cgcaccgaca atgcccttaa aaatcactgg 480catgtcctaa tggctaggaa
gatgcgtcag cagagctcta gctacgttca gcgcttcaac 540ggaagcgcac
atgagtctaa caccgatcat aagatcttta accttagccc cggtaacgtg
600gacgacgatg aggatgttaa ccttaaaaag tgtagttggg agatgcttaa
agagggcact 660actaacctta aggctcaata tcttcaggag gaatattcta
gctcgcggat gccgatgcag 720ggaccgcacc accattactc taccttccca
gctgattctc tagctctcac gctacacgtg 780tcaattcaag agcctagcag
ttctagtagc ttaagcctac cttcctcttc aactacaggt 840gagcacacta
tggtcactag atatttcgag actattaagc cccccgcctt tatagacttt
900ttgggcgttg gtcactaa 91823918DNAArtificial SequenceSynthetic
polynucleotide 23atgaagatgg actttagctg ctttcaagag taccccttcg
agtttcactg taggggcact 60acctttaacg gctttagaga gaacaatgcc gtttcagaga
ctgtggagga gttctgtaac 120aaacgtagga tgcaaaagaa gtccgacgac
cttaaaacta agaagaagaa gaagcagtca 180gttagtaggg tgtgctctag
gggccattgg cgtatttctg aggataccca gctaatggaa 240ctagtttcag
tctacggccc gcagaactgg aatcatattg ccgaatctat gcagggtagg
300accggtaagt cttgtcgtct tcgttggttt aaccagctag accctaggat
aaacaagcgc 360gccttcagtg acgaggagga agagagacta ctagccgccc
atcgggcttt cggtaacaag 420tgggcgatga tagctaagct ctttaacggt
aggaccgata acgcccttaa gaatcactgg 480catgtgctaa tggcaaggaa
gatgaggcaa cagagttcta gctatgttca gcgctttaac 540ggatcagctc
acgagtctaa caccgatcac aagatcttta accttagccc cggtaatgtg
600gacgacgacg aggacgtgaa ccttaaaaag tgcagttggg agatgcttaa
agaaggcact 660actaacctta aggctcaata cttacaagaa gagtattctt
cgtctaggat gcctatgcag 720ggacctcacc accactattc taccttccca
gctgatagcc tagctctaac ccttcacgtt 780agtattcaag agcctagctc
ctcgagtagc ctgtccctac cttccagttc aactaccggt 840gaacacacta
tggtcactag atacttcgaa acgattaaac ccccagcctt tattgatttt
900ctaggcgttg gtcactaa 91824918DNAArtificial SequenceSynthetic
polynucleotide 24atgaagatgg actttagctg ctttcaagag tacccatttg
agttccactg taggggcact 60accttcaacg gctttagaga gaacaacgcc gttagcgaga
ctgtggaaga attctgtaat 120aagcggagga tgcagaagaa gtcagacgac
cttaagacca agaagaagaa gaaacagtca 180gttagtaggg tgtgctctag
gggtcactgg cgtataagtg aagatactca gctaatggaa 240ctagtttcag
tctacggccc tcagaactgg aatcatatag cagagtctat gcaaggtagg
300accggtaagt cttgtaggct tcgttggttt aatcagctcg accctaggat
taacaagcgc 360gcctttagtg acgaagagga agagagacta ctagccgctc
acagagcttt cggtaacaag 420tgggctatga tagctaagct ctttaacggt
aggaccgata acgcccttaa gaatcactgg 480catgttctaa tggcgaggaa
gatgaggcag cagagttcta gctacgttca gcgctttaac 540ggatctgctc
acgagtctaa cacagaccac aagatcttta accttagccc cggtaatgtg
600gacgacgacg aggacgttaa tcttaaaaag tgcagttggg agatgcttaa
agagggcact 660actaacctta aggctcagta ccttcaagaa gagtactcta
gctctaggat gcctatgcaa 720ggaccgcacc accactactc tacgttccca
gctgatagcc tagctctaac ccttcacgtt 780agtattcaag agcctagcag
ttctagtagc ctgagcctac ctagcagttc aactaccggt 840gagcacacta
tggtcactag atacttcgag actattaagc ccccagcctt tatagatttt
900ctaggcgttg gtcactaa 91825699DNAArtificial SequenceSynthetic
polynucleotide 25atggactggt gcagcttcaa cgagtggccc ttcgagtacc
acagcagaat cacctgctgg 60aacatgtaca gagacaacca ggccctgacc gactgcgccg
aggagttcag ccagagaaga 120cacatgaaca gaaagaccga cgagatcaag
acccacagaa gaagaaagca gagcgccagc 180agagtgtgca gcagaggcca
ctggagaatc agcgaggaca cccagctgat ggagctggtg 240agcgtgtacg
gcccccagaa ctggaaccac atcgccgaga gcatgcaggg cagaaccggc
300aagagctgca gactgagatg gttcaaccag ctggacccca gaatcaacaa
gagagccttc 360agcgacgagg aggaggagag actgctggcc gcccacagag
ccttcggcaa caagtgggcc 420atgatcgcca agctgttcaa cggcagaacc
gacaacgccc tgaagaacca ctggcacgtg 480ctgatggcca gaaagatgag
acagcagagc agcagctgga tcaacaagta ccagatcagc 540gccaaggaca
gcaacaccga gcacaagggc ttccagctga gccccatgat caccgccctg
600tgcgtgaagc tgtgcgtgga cttccagagc gccgccgtgg ccagattctt
caagttcctg 660agcgtgagaa accaggagct gatcatgtac agcaacaga
69926233PRTArtificial SequenceAmino acid sequence MybTF, variant 17
26Met Asp Trp Cys Ser Phe Asn Glu Trp Pro Phe Glu Tyr His Ser Arg 1
5 10 15 Ile Thr Cys Trp Asn Met Tyr Arg Asp Asn Gln Ala Leu Thr Asp
Cys 20 25 30 Ala Glu Glu Phe Ser Gln Arg Arg His Met Asn Arg Lys
Thr Asp Glu 35 40 45 Ile Lys Thr His Arg Arg Arg Lys Gln Ser Ala
Ser Arg Val Cys Ser 50 55 60 Arg Gly His Trp Arg Ile Ser Glu Asp
Thr Gln Leu Met Glu Leu Val 65 70 75 80 Ser Val Tyr Gly Pro Gln Asn
Trp Asn His Ile Ala Glu Ser Met Gln 85 90 95 Gly Arg Thr Gly Lys
Ser Cys Arg Leu Arg Trp Phe Asn Gln Leu Asp 100 105 110 Pro Arg Ile
Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu Arg Leu 115 120 125 Leu
Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile Ala Lys 130 135
140 Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp His Val
145 150 155 160 Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Trp
Ile Asn Lys 165 170 175 Tyr Gln Ile Ser Ala Lys Asp Ser Asn Thr Glu
His Lys Gly Phe Gln 180 185 190 Leu Ser Pro Met Ile Thr Ala Leu Cys
Val Lys Leu Cys Val Asp Phe 195 200 205 Gln Ser Ala Ala Val Ala Arg
Phe Phe Lys Phe Leu Ser Val Arg Asn 210 215 220 Gln Glu Leu Ile Met
Tyr Ser Asn Arg 225 230 27699DNAArtificial SequenceSynthetic
polynucleotide 27atggacttca gcacctggca ggagtggccc ttcgagtacc
acagcaaggg caccagctgg 60aacggctaca gagaccagaa catcatgtgc gacaccgtgg
acgactactg caacaagaga 120cacgcccaga agaagaccga ggaggccaga
acccacaaga agaagcacca gagcgccagc 180agagtgtgca gcagaggcca
ctggagaatc agcgaggaca cccagctgat ggagctggtg 240agcgtgtacg
gcccccagaa ctggaaccac atcgccgaga gcatgcaggg cagaaccggc
300aagagctgca gactgagatg gttcaaccag ctggacccca gaatcaacaa
gagagccttc 360agcgacgagg aggaggagag actgctggcc gcccacagag
ccttcggcaa caagtgggcc 420atgatcgcca agctgttcaa cggcagaacc
gacaacgccc tgaagaacca ctggcacgtg 480ctgatggcca gaaagatgag
acagcagagc agctgctacg tgaacagatt caacctgagc 540gccaaggaga
gccagaccga cagacacatg tggaacgcca cccccctgct gtgcggcatc
600agcctgaagg ccagcatcga ctacaactgc gtgatcatca tcagatggtt
cagattcctg 660agcctgcacc agaacgacct gatggtgtgg agcaacaga
69928233PRTArtificial SequenceAmino acid sequence MybTF, variant 18
28Met Asp Phe Ser Thr Trp Gln Glu Trp Pro Phe Glu Tyr His Ser Lys 1
5 10 15 Gly Thr Ser Trp Asn Gly Tyr Arg Asp Gln Asn Ile Met Cys Asp
Thr 20 25 30 Val Asp Asp Tyr Cys Asn Lys Arg His Ala Gln Lys Lys
Thr Glu Glu 35 40 45 Ala Arg Thr His Lys Lys Lys His Gln Ser Ala
Ser Arg Val Cys Ser 50 55 60 Arg Gly His Trp Arg Ile Ser Glu Asp
Thr Gln Leu Met Glu Leu Val 65 70 75 80 Ser Val Tyr Gly Pro Gln Asn
Trp Asn His Ile Ala Glu Ser Met Gln 85 90 95 Gly Arg Thr Gly Lys
Ser Cys Arg Leu Arg Trp Phe Asn Gln Leu Asp 100 105 110 Pro Arg Ile
Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu Arg Leu 115 120 125 Leu
Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile Ala Lys 130 135
140 Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp His Val
145 150 155 160 Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser Cys Tyr
Val Asn Arg 165 170 175 Phe Asn Leu Ser Ala Lys Glu Ser Gln Thr Asp
Arg His Met Trp Asn 180 185 190 Ala Thr Pro Leu Leu Cys Gly Ile Ser
Leu Lys Ala Ser Ile Asp Tyr 195 200 205 Asn Cys Val Ile Ile Ile Arg
Trp Phe Arg Phe Leu Ser Leu His Gln 210 215 220 Asn Asp Leu Met Val
Trp Ser Asn Arg 225 230 29699DNAArtificial SequenceSynthetic
polynucleotide 29atggactaca ccacctggaa cgactggccc tgggactacc
actgccacgg caccaccttc 60aacatgttcc acgagaacaa cgccgtgacc gagtgcgtgg
aggagttctg ccagcaccac 120agaatgcaga agaagagcga ggagctgaag
accagaagaa agcacaagaa cagcgtgagc 180agagtgtgca gcagaggcca
ctggagaatc agcgaggaca cccagctgat ggagctggtg 240agcgtgtacg
gcccccagaa ctggaaccac atcgccgaga gcatgcaggg cagaaccggc
300aagagctgca gactgagatg gttcaaccag ctggacccca gaatcaacaa
gagagccttc 360agcgacgagg aggaggagag actgctggcc gcccacagag
ccttcggcaa caagtgggcc 420atgatcgcca agctgttcaa cggcagaacc
gacaacgccc tgaagaacca ctggcacgtg 480ctgatggcca gaaagatgag
acagcagagc agcagctacg tgcagagatt caacggctgc 540atgcacgaga
ccaactgcga caagaaggcc
tggaacctga gccccgtgct gaccctgctg 600accggcaagg gcagcatcga
cttccagagc gtgatcgcca tgagattctt caagtacctg 660agcctgagaa
accaggagct ggtgctgtac agccagaag 69930233PRTArtificial SequenceAmino
acid sequence MybTF, variant 19 30Met Asp Tyr Thr Thr Trp Asn Asp
Trp Pro Trp Asp Tyr His Cys His 1 5 10 15 Gly Thr Thr Phe Asn Met
Phe His Glu Asn Asn Ala Val Thr Glu Cys 20 25 30 Val Glu Glu Phe
Cys Gln His His Arg Met Gln Lys Lys Ser Glu Glu 35 40 45 Leu Lys
Thr Arg Arg Lys His Lys Asn Ser Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val Gln Arg 165 170 175 Phe
Asn Gly Cys Met His Glu Thr Asn Cys Asp Lys Lys Ala Trp Asn 180 185
190 Leu Ser Pro Val Leu Thr Leu Leu Thr Gly Lys Gly Ser Ile Asp Phe
195 200 205 Gln Ser Val Ile Ala Met Arg Phe Phe Lys Tyr Leu Ser Leu
Arg Asn 210 215 220 Gln Glu Leu Val Leu Tyr Ser Gln Lys 225 230
31699DNAArtificial SequenceSynthetic polynucleotide 31atggactaca
gcaccttcca ggactacccc ttcgagttca agtgcagagg caccaccttc 60cagggctaca
gagagcagaa cgccgtgagc gagagcgtgg aggagttctg caacaagaga
120agaatgcagc acaagagcga ggacctgaga accaagcaca gaagaaagaa
cagcgtgagc 180agagtgtgca gcagaggcca ctggagaatc agcgaggaca
cccagctgat ggagctggtg 240agcgtgtacg gcccccagaa ctggaaccac
atcgccgaga gcatgcaggg cagaaccggc 300aagagctgca gactgagatg
gttcaaccag ctggacccca gaatcaacaa gagagccttc 360agcgacgagg
aggaggagag actgctggcc gcccacagag ccttcggcaa caagtgggcc
420atgatcgcca agctgttcaa cggcagaacc gacaacgccc tgaagaacca
ctggcacgtg 480ctgatggcca gaaagatgag acagcagagc agctgctacg
gcaacagatt caacatgacc 540atccacgaga gcaacagcga cagaaagatc
tacaacctga gccccggcct gtgcctgctg 600agcctgcaca tcaccatcga
gtggcagtgc gtgatcgtga tgagatactt cagatgggcc 660accctgagaa
acaacgagct gctggtgttc agccagaag 69932233PRTArtificial SequenceAmino
acid sequence MybTF, variant 20 32Met Asp Tyr Ser Thr Phe Gln Asp
Tyr Pro Phe Glu Phe Lys Cys Arg 1 5 10 15 Gly Thr Thr Phe Gln Gly
Tyr Arg Glu Gln Asn Ala Val Ser Glu Ser 20 25 30 Val Glu Glu Phe
Cys Asn Lys Arg Arg Met Gln His Lys Ser Glu Asp 35 40 45 Leu Arg
Thr Lys His Arg Arg Lys Asn Ser Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Cys Tyr Gly Asn Arg 165 170 175 Phe
Asn Met Thr Ile His Glu Ser Asn Ser Asp Arg Lys Ile Tyr Asn 180 185
190 Leu Ser Pro Gly Leu Cys Leu Leu Ser Leu His Ile Thr Ile Glu Trp
195 200 205 Gln Cys Val Ile Val Met Arg Tyr Phe Arg Trp Ala Thr Leu
Arg Asn 210 215 220 Asn Glu Leu Leu Val Phe Ser Gln Lys 225 230
33699DNAArtificial SequenceSynthetic polynucleotide 33atggacttca
gctgctggaa cgagtacccc ttcgagttca gatgcagaat caccagcttc 60aacgcctgga
gagagcagca ggccgtgagc gacaccgtgg aggagttctg caacaagaga
120cacatgaaca agaagtgcga cgacctgaag accaagaaga agaagaagca
gagcgtgagc 180agagtgtgca gcagaggcca ctggagaatc agcgaggaca
cccagctgat ggagctggtg 240agcgtgtacg gcccccagaa ctggaaccac
atcgccgaga gcatgcaggg cagaaccggc 300aagagctgca gactgagatg
gttcaaccag ctggacccca gaatcaacaa gagagccttc 360agcgacgagg
aggaggagag actgctggcc gcccacagag ccttcggcaa caagtgggcc
420atgatcgcca agctgttcaa cggcagaacc gacaacgccc tgaagaacca
ctggcacgtg 480ctgatggcca gaaagatgag acagcagagc agcagctacg
tgcagagatt caacgtgagc 540ctgcacgaga gccagaccga gcacagaatc
ttcaacggca gccccggcct gagcctgctg 600tgcctgcaca tctgcatcga
gttcaacacc gtgatcgtga tgagatactt ccactacctg 660agcctgagaa
acaacgagat catggtgtgg agccagaag 69934233PRTArtificial SequenceAmino
acid sequence MybTF, variant 21 34Met Asp Phe Ser Cys Trp Asn Glu
Tyr Pro Phe Glu Phe Arg Cys Arg 1 5 10 15 Ile Thr Ser Phe Asn Ala
Trp Arg Glu Gln Gln Ala Val Ser Asp Thr 20 25 30 Val Glu Glu Phe
Cys Asn Lys Arg His Met Asn Lys Lys Cys Asp Asp 35 40 45 Leu Lys
Thr Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val Gln Arg 165 170 175 Phe
Asn Val Ser Leu His Glu Ser Gln Thr Glu His Arg Ile Phe Asn 180 185
190 Gly Ser Pro Gly Leu Ser Leu Leu Cys Leu His Ile Cys Ile Glu Phe
195 200 205 Asn Thr Val Ile Val Met Arg Tyr Phe His Tyr Leu Ser Leu
Arg Asn 210 215 220 Asn Glu Ile Met Val Trp Ser Gln Lys 225 230
35699DNAArtificial SequenceSynthetic polynucleotide 35atggacttct
gctgctacca ggagtggccc ttcgagttca gatgcagagg caccaccttc 60aacggcttca
gagagaacaa cctggtgagc gacaccggcg aggagttctg caacaagaga
120agaatgcaga agagaagcga cgacctgaga agcaagaaga agaagaagca
gagcgtgagc 180agagtgtgca gcagaggcca ctggagaatc agcgaggaca
cccagctgat ggagctggtg 240agcgtgtacg gcccccagaa ctggaaccac
atcgccgaga gcatgcaggg cagaaccggc 300aagagctgca gactgagatg
gttcaaccag ctggacccca gaatcaacaa gagagccttc 360agcgacgagg
aggaggagag actgctggcc gcccacagag ccttcggcaa caagtgggcc
420atgatcgcca agctgttcaa cggcagaacc gacaacgccc tgaagaacca
ctggcacgtg 480ctgatggcca gaaagatgag acagcagagc agcagctacg
tgcagagatt caacggcagc 540gcccacgaga gcaacaccga ccacaagatc
ttcaacgcca gccccggcct gagcggcctg 600accctgcacc tgtgcatcga
gttcaacagc gtgatcgtga tgagattctg gagatacctg 660agcctgagac
agcaggagat gatggtgtgg agccagaag 69936233PRTArtificial SequenceAmino
acid sequence MybTF, variant 22 36Met Asp Phe Cys Cys Tyr Gln Glu
Trp Pro Phe Glu Phe Arg Cys Arg 1 5 10 15 Gly Thr Thr Phe Asn Gly
Phe Arg Glu Asn Asn Leu Val Ser Asp Thr 20 25 30 Gly Glu Glu Phe
Cys Asn Lys Arg Arg Met Gln Lys Arg Ser Asp Asp 35 40 45 Leu Arg
Ser Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val Gln Arg 165 170 175 Phe
Asn Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile Phe Asn 180 185
190 Ala Ser Pro Gly Leu Ser Gly Leu Thr Leu His Leu Cys Ile Glu Phe
195 200 205 Asn Ser Val Ile Val Met Arg Phe Trp Arg Tyr Leu Ser Leu
Arg Gln 210 215 220 Gln Glu Met Met Val Trp Ser Gln Lys 225 230
37699DNAArtificial SequenceSynthetic polynucleotide 37atggacttca
gctgcttcca ggagtacccc ttcgagttcc acaccaaggg caccaccttc 60aacggcttca
gagagaacaa cgccggcacc gagaccgtgg aggagttctg caacaagaga
120agactgcaga agaagagcga cgacctgaag accaagaaga agaagaagca
gagcgtgagc 180agagtgtgca gcagaggcca ctggagaatc agcgaggaca
cccagctgat ggagctggtg 240agcgtgtacg gcccccagaa ctggaaccac
atcgccgaga gcatgcaggg cagaaccggc 300aagagctgca gactgagatg
gttcaaccag ctggacccca gaatcaacaa gagagccttc 360agcgacgagg
aggaggagag actgctggcc gcccacagag ccttcggcaa caagtgggcc
420atgatcgcca agctgttcaa cggcagaacc gacaacgccc tgaagaacca
ctggcacgtg 480ctgatggcca gaaagatgag acagcagagc agcagctacg
tgaacagatt ccagggcagc 540gcccacgaga gcaacaccga ccacaagatc
tggaacctga gccccggcct gagcctgctg 600accctgcaca tctgcatcga
gttcaactgc gtgatcgtga tgagatactt cagatacctg 660tgcctgagaa
acaacgacct gatggtgtgg agccagaag 69938233PRTArtificial SequenceAmino
acid sequence MybTF, variant 23 38Met Asp Phe Ser Cys Phe Gln Glu
Tyr Pro Phe Glu Phe His Thr Lys 1 5 10 15 Gly Thr Thr Phe Asn Gly
Phe Arg Glu Asn Asn Ala Gly Thr Glu Thr 20 25 30 Val Glu Glu Phe
Cys Asn Lys Arg Arg Leu Gln Lys Lys Ser Asp Asp 35 40 45 Leu Lys
Thr Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val Asn Arg 165 170 175 Phe
Gln Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile Trp Asn 180 185
190 Leu Ser Pro Gly Leu Ser Leu Leu Thr Leu His Ile Cys Ile Glu Phe
195 200 205 Asn Cys Val Ile Val Met Arg Tyr Phe Arg Tyr Leu Cys Leu
Arg Asn 210 215 220 Asn Asp Leu Met Val Trp Ser Gln Lys 225 230
39699DNAArtificial SequenceSynthetic polynucleotide 39atggacttca
gctgcttcca ggagtacccc ttcgagttcc actgcagagg caccaccttc 60aacggcttca
gagacaacaa cgccgtgagc gagagcgtgg aggagttctg caacaagaga
120agaatgcaga agaagagcga cgacctgaag accaagaaga agcacaagca
gaccgtgagc 180agagtgtgca gcagaggcca ctggagaatc agcgaggaca
cccagctgat ggagctggtg 240agcgtgtacg gcccccagaa ctggaaccac
atcgccgaga gcatgcaggg cagaaccggc 300aagagctgca gactgagatg
gttcaaccag ctggacccca gaatcaacaa gagagccttc 360agcgacgagg
aggaggagag actgctggcc gcccacagag ccttcggcaa caagtgggcc
420atgatcgcca agctgttcaa cggcagaacc gacaacgccc tgaagaacca
ctggcacgtg 480ctgatggcca gaaagatgag acagcagagc agcagctacg
tgcagagatt caacggcagc 540gcccacgaga gcaacaccga ccacaagatc
ttcaacctgt gccccggcct gagcctgctg 600accctgcaca tctgcatcga
gttcaacagc gtgatcgtga tgagatactg gagatacctg 660agcctgagaa
acaacgagct gatggtgtgg agccagaag 69940233PRTArtificial SequenceAmino
acid sequence MybTF, variant 24 40Met Asp Phe Ser Cys Phe Gln Glu
Tyr Pro Phe Glu Phe His Cys Arg 1 5 10 15 Gly Thr Thr Phe Asn Gly
Phe Arg Asp Asn Asn Ala Val Ser Glu Ser 20 25 30 Val Glu Glu Phe
Cys Asn Lys Arg Arg Met Gln Lys Lys Ser Asp Asp 35 40 45 Leu Lys
Thr Lys Lys Lys His Lys Gln Thr Val Ser Arg Val Cys Ser 50 55 60
Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu Leu Val 65
70 75 80 Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu Ser
Met Gln 85 90 95 Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp Phe
Asn Gln Leu Asp 100 105 110 Pro Arg Ile Asn Lys Arg Ala Phe Ser Asp
Glu Glu Glu Glu Arg Leu 115 120 125 Leu Ala Ala His Arg Ala Phe Gly
Asn Lys Trp Ala Met Ile Ala Lys 130 135 140 Leu Phe Asn Gly Arg Thr
Asp Asn Ala Leu Lys Asn His Trp His Val 145 150 155 160 Leu Met Ala
Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val Gln Arg 165 170 175 Phe
Asn Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile Phe Asn 180 185
190 Leu Cys Pro Gly Leu Ser Leu Leu Thr Leu His Ile Cys Ile Glu Phe
195 200 205 Asn Ser Val Ile Val Met Arg Tyr Trp Arg Tyr Leu Ser Leu
Arg Asn 210 215 220 Asn Glu Leu Met Val Trp Ser Gln Lys 225 230
41915DNAArtificial SequenceSynthetic polynucleotide 41atgagaggcg
actggtgcag ctacaacgac ttcccctggg agttcaagac ccacatgagc 60acctggaaca
tgtggagaga gaacaacgcc gtgtgcgaga ccgtggagga ctactgccag
120aagcaccaca tcaacaagaa gtgcgaggac atcaagaccc acaagaagca
caagaacagc 180gtgagcagag tgtgcagcag aggccactgg agaatcagcg
aggacaccca gctgatggag 240ctggtgagcg tgtacggccc ccagaactgg
aaccacatcg ccgagagcat gcagggcaga 300accggcaaga gctgcagact
gagatggttc aaccagctgg accccagaat caacaagaga 360gccttcagcg
acgaggagga ggagagactg ctggccgccc acagagcctt cggcaacaag
420tgggccatga tcgccaagct gttcaacggc agaaccgaca acgccctgaa
gaaccactgg 480cacgtgctga tggccagaaa gatgagacag cagagcagca
gctacgtgca gagattcaac 540ggcaccgcca gagagagcaa caccgagcac
aagatcttca acctgagccc cgccaacgtg 600gaggacgagg aggacggcca
gatgcaccac accaccttcg acatcgtgag agacggcacc 660agcaacctga
aggccaacta cctgcaggag gagtacacct gcacccacgc ccccctgcag
720ggccccaaga agaagttcag cagctggccc gccgagtgcc tggtgatcac
cgcccacatc 780agcatcaacg accccagctg cagcagcagc atcagcctgc
cctgctgctg caccaccggc 840gagaagacca tgctgtgcag atacttcgac
accatcaagc cccccatgtt cctggactgg 900ctgggcctgg gcaga
91542305PRTArtificial SequenceAmino acid sequence MybTF, variant 25
42Met Arg Gly Asp Trp Cys Ser Tyr Asn Asp Phe Pro Trp Glu Phe Lys 1
5 10 15 Thr His Met Ser Thr Trp Asn Met Trp Arg Glu Asn Asn Ala Val
Cys 20 25 30 Glu Thr Val Glu Asp Tyr Cys Gln Lys His His Ile Asn
Lys Lys Cys 35 40 45 Glu Asp Ile Lys Thr His Lys Lys His Lys Asn
Ser Val Ser Arg Val 50 55 60 Cys Ser Arg Gly His Trp Arg Ile Ser
Glu Asp
Thr Gln Leu Met Glu 65 70 75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn
Trp Asn His Ile Ala Glu Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys
Ser Cys Arg Leu Arg Trp Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile
Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu
Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala
Lys Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp 145 150
155 160 His Val Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr
Val 165 170 175 Gln Arg Phe Asn Gly Thr Ala Arg Glu Ser Asn Thr Glu
His Lys Ile 180 185 190 Phe Asn Leu Ser Pro Ala Asn Val Glu Asp Glu
Glu Asp Gly Gln Met 195 200 205 His His Thr Thr Phe Asp Ile Val Arg
Asp Gly Thr Ser Asn Leu Lys 210 215 220 Ala Asn Tyr Leu Gln Glu Glu
Tyr Thr Cys Thr His Ala Pro Leu Gln 225 230 235 240 Gly Pro Lys Lys
Lys Phe Ser Ser Trp Pro Ala Glu Cys Leu Val Ile 245 250 255 Thr Ala
His Ile Ser Ile Asn Asp Pro Ser Cys Ser Ser Ser Ile Ser 260 265 270
Leu Pro Cys Cys Cys Thr Thr Gly Glu Lys Thr Met Leu Cys Arg Tyr 275
280 285 Phe Asp Thr Ile Lys Pro Pro Met Phe Leu Asp Trp Leu Gly Leu
Gly 290 295 300 Arg 305 43915DNAArtificial SequenceSynthetic
polynucleotide 43atgaagatgg acttctgcag ctacaacgag tggcccttcg
agttcaagag ccacggcagc 60acctacaacg cctggaagga caacaacgcc gtgtgcgact
gcgtggagga cttctgcaac 120aagcacaagc tgcagaagag aagcgacgac
gccaagacca agaagaagca caagaacagc 180gtgagcagag tgtgcagcag
aggccactgg agaatcagcg aggacaccca gctgatggag 240ctggtgagcg
tgtacggccc ccagaactgg aaccacatcg ccgagagcat gcagggcaga
300accggcaaga gctgcagact gagatggttc aaccagctgg accccagaat
caacaagaga 360gccttcagcg acgaggagga ggagagactg ctggccgccc
acagagcctt cggcaacaag 420tgggccatga tcgccaagct gttcaacggc
agaaccgaca acgccctgaa gaaccactgg 480cacgtgctga tggccagaaa
gatgagacag cagagcagca gctacgtgca gagattcaac 540ggcaccctgc
acgacaccaa caccgaccac cacgtgttcc agggcagccc cggcaacgtg
600gaggacgacg acgacgtgaa cgtgagaaag tgctgctggg agatcatcaa
ggagggcagc 660acccagctga gagcccagtg ggcccaggac gagtacagca
gcaccaaggg ccccatgcag 720ggcccccaca gaaagtacag ctgcttcccc
atcgagagca tggccctgag cctgcacgtg 780accctgcagg accccagcag
caccagcacc ggcaccctgc ccagcacctg caccaccgtg 840gagcacagca
tggtgagcag atacttcgag accatcaagc cccccctgtt catcgactac
900gtgggcgtgg gccac 91544305PRTArtificial SequenceAmino acid
sequence MybTF, variant 26 44Met Lys Met Asp Phe Cys Ser Tyr Asn
Glu Trp Pro Phe Glu Phe Lys 1 5 10 15 Ser His Gly Ser Thr Tyr Asn
Ala Trp Lys Asp Asn Asn Ala Val Cys 20 25 30 Asp Cys Val Glu Asp
Phe Cys Asn Lys His Lys Leu Gln Lys Arg Ser 35 40 45 Asp Asp Ala
Lys Thr Lys Lys Lys His Lys Asn Ser Val Ser Arg Val 50 55 60 Cys
Ser Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu 65 70
75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala Glu
Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg Trp
Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile Asn Lys Arg Ala Phe Ser
Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu Ala Ala His Arg Ala Phe
Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala Lys Leu Phe Asn Gly Arg
Thr Asp Asn Ala Leu Lys Asn His Trp 145 150 155 160 His Val Leu Met
Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val 165 170 175 Gln Arg
Phe Asn Gly Thr Leu His Asp Thr Asn Thr Asp His His Val 180 185 190
Phe Gln Gly Ser Pro Gly Asn Val Glu Asp Asp Asp Asp Val Asn Val 195
200 205 Arg Lys Cys Cys Trp Glu Ile Ile Lys Glu Gly Ser Thr Gln Leu
Arg 210 215 220 Ala Gln Trp Ala Gln Asp Glu Tyr Ser Ser Thr Lys Gly
Pro Met Gln 225 230 235 240 Gly Pro His Arg Lys Tyr Ser Cys Phe Pro
Ile Glu Ser Met Ala Leu 245 250 255 Ser Leu His Val Thr Leu Gln Asp
Pro Ser Ser Thr Ser Thr Gly Thr 260 265 270 Leu Pro Ser Thr Cys Thr
Thr Val Glu His Ser Met Val Ser Arg Tyr 275 280 285 Phe Glu Thr Ile
Lys Pro Pro Leu Phe Ile Asp Tyr Val Gly Val Gly 290 295 300 His 305
45915DNAArtificial SequenceSynthetic polynucleotide 45atgaagatgg
actggagctg cttcaacgag ttccccttcg actacagaac cagactgtgc 60accttcaacg
ccttcagaga gcagaacgcc gtgagcgaga gcgtggagga ctggtgcaac
120aagagaagaa tgcagcacca caccgaggac gtgagaacca agagaaagag
aaagcagagc 180gtgagcagag tgtgcagcag aggccactgg agaatcagcg
aggacaccca gctgatggag 240ctggtgagcg tgtacggccc ccagaactgg
aaccacatcg ccgagagcat gcagggcaga 300accggcaaga gctgcagact
gagatggttc aaccagctgg accccagaat caacaagaga 360gccttcagcg
acgaggagga ggagagactg ctggccgccc acagagcctt cggcaacaag
420tgggccatga tcgccaagct gttcaacggc agaaccgaca acgccctgaa
gaaccactgg 480cacgtgctga tggccagaaa gatgagacag cagagcagca
gctacgtgca gagattcaac 540ggcagcgccc acgagagcaa ctgcgagcac
agagccttca acctgagccc cctgaacgtg 600gaggaggacg acgacggcca
gatgaagaag tgcagctggg agatgctgaa ggacggcacc 660acccaggcca
agctgcagtt cctgaacgag gactacagct gcagcagagt gcccgcccag
720ggcccccaca gacactggag caccttcccc gccgacagcg ccgccgtgac
cctgaaggtg 780agcatcaacg agcccagcac cagcaccagc ctgagcatcc
cctgcagcag cagcaccgcc 840gagcacacca tggtgaccag attcttcgag
accatcaagc cccccgcctt catcgacttc 900ctgggcgtgg gcaga
91546305PRTArtificial SequenceAmino acid sequence MybTF, variant 27
46Met Lys Met Asp Trp Ser Cys Phe Asn Glu Phe Pro Phe Asp Tyr Arg 1
5 10 15 Thr Arg Leu Cys Thr Phe Asn Ala Phe Arg Glu Gln Asn Ala Val
Ser 20 25 30 Glu Ser Val Glu Asp Trp Cys Asn Lys Arg Arg Met Gln
His His Thr 35 40 45 Glu Asp Val Arg Thr Lys Arg Lys Arg Lys Gln
Ser Val Ser Arg Val 50 55 60 Cys Ser Arg Gly His Trp Arg Ile Ser
Glu Asp Thr Gln Leu Met Glu 65 70 75 80 Leu Val Ser Val Tyr Gly Pro
Gln Asn Trp Asn His Ile Ala Glu Ser 85 90 95 Met Gln Gly Arg Thr
Gly Lys Ser Cys Arg Leu Arg Trp Phe Asn Gln 100 105 110 Leu Asp Pro
Arg Ile Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu 115 120 125 Arg
Leu Leu Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile 130 135
140 Ala Lys Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp
145 150 155 160 His Val Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser
Ser Tyr Val 165 170 175 Gln Arg Phe Asn Gly Ser Ala His Glu Ser Asn
Cys Glu His Arg Ala 180 185 190 Phe Asn Leu Ser Pro Leu Asn Val Glu
Glu Asp Asp Asp Gly Gln Met 195 200 205 Lys Lys Cys Ser Trp Glu Met
Leu Lys Asp Gly Thr Thr Gln Ala Lys 210 215 220 Leu Gln Phe Leu Asn
Glu Asp Tyr Ser Cys Ser Arg Val Pro Ala Gln 225 230 235 240 Gly Pro
His Arg His Trp Ser Thr Phe Pro Ala Asp Ser Ala Ala Val 245 250 255
Thr Leu Lys Val Ser Ile Asn Glu Pro Ser Thr Ser Thr Ser Leu Ser 260
265 270 Ile Pro Cys Ser Ser Ser Thr Ala Glu His Thr Met Val Thr Arg
Phe 275 280 285 Phe Glu Thr Ile Lys Pro Pro Ala Phe Ile Asp Phe Leu
Gly Val Gly 290 295 300 Arg 305 47915DNAArtificial
SequenceSynthetic polynucleotide 47atgcacatgg acttcagctg cttccaggag
ttcccctacg agtggcactg cagagtgacc 60accttcaacg gcttccacga caacaacgcc
gtgagcgaga ccgtggagga gttctgcaac 120aagagaagaa tgcagaagaa
gagcgacgag ctgagaacca agaagaagaa gaagaacagc 180gtgagcagag
tgtgcagcag aggccactgg agaatcagcg aggacaccca gctgatggag
240ctggtgagcg tgtacggccc ccagaactgg aaccacatcg ccgagagcat
gcagggcaga 300accggcaaga gctgcagact gagatggttc aaccagctgg
accccagaat caacaagaga 360gccttcagcg acgaggagga ggagagactg
ctggccgccc acagagcctt cggcaacaag 420tgggccatga tcgccaagct
gttcaacggc agaaccgaca acgccctgaa gaaccactgg 480cacgtgctga
tggccagaaa gatgagacag cagagcagca gctacgtgca gagattcaac
540ggcagcgccc acgagagcaa ctgcgacaga cacatcttca acctgacccc
cggcaacgtg 600gaggacgacg aggacgtgaa cctgaagcac tgcagcttcg
acatcgtgaa ggagggcacc 660tgcaacggca aggcccagta cggccaggag
gactacagca gctgcagaat gcccatgaac 720ggcccccacc accactacag
caccttcccc gccgacaccc tggccgccac cgcccacgtg 780tgcatccagg
agcccagcag ctgcagcacc gtgagcctgc ccagcagcag caccaccggc
840gaccacaccg gcgtgaccca ctacttcgag agcatcagac cccccgcctt
catcgactac 900ctggccgtgg gcaga 91548305PRTArtificial SequenceAmino
acid sequence MybTF, variant 28 48Met His Met Asp Phe Ser Cys Phe
Gln Glu Phe Pro Tyr Glu Trp His 1 5 10 15 Cys Arg Val Thr Thr Phe
Asn Gly Phe His Asp Asn Asn Ala Val Ser 20 25 30 Glu Thr Val Glu
Glu Phe Cys Asn Lys Arg Arg Met Gln Lys Lys Ser 35 40 45 Asp Glu
Leu Arg Thr Lys Lys Lys Lys Lys Asn Ser Val Ser Arg Val 50 55 60
Cys Ser Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu 65
70 75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala
Glu Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg
Trp Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile Asn Lys Arg Ala Phe
Ser Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu Ala Ala His Arg Ala
Phe Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala Lys Leu Phe Asn Gly
Arg Thr Asp Asn Ala Leu Lys Asn His Trp 145 150 155 160 His Val Leu
Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val 165 170 175 Gln
Arg Phe Asn Gly Ser Ala His Glu Ser Asn Cys Asp Arg His Ile 180 185
190 Phe Asn Leu Thr Pro Gly Asn Val Glu Asp Asp Glu Asp Val Asn Leu
195 200 205 Lys His Cys Ser Phe Asp Ile Val Lys Glu Gly Thr Cys Asn
Gly Lys 210 215 220 Ala Gln Tyr Gly Gln Glu Asp Tyr Ser Ser Cys Arg
Met Pro Met Asn 225 230 235 240 Gly Pro His His His Tyr Ser Thr Phe
Pro Ala Asp Thr Leu Ala Ala 245 250 255 Thr Ala His Val Cys Ile Gln
Glu Pro Ser Ser Cys Ser Thr Val Ser 260 265 270 Leu Pro Ser Ser Ser
Thr Thr Gly Asp His Thr Gly Val Thr His Tyr 275 280 285 Phe Glu Ser
Ile Arg Pro Pro Ala Phe Ile Asp Tyr Leu Ala Val Gly 290 295 300 Arg
305 49915DNAArtificial SequenceSynthetic polynucleotide
49atgaagatgg actacagctg cttccaggag taccccttcg acttccactg cagagccacc
60accttcaacg gcttccacga gaacaacgcc gtgagcgaga ccgtggagga gttctgcaac
120cacagaagaa tgcagaagaa gagcgacgac ggccacacca agagaaagaa
gagacagagc 180gtgagcagag tgtgcagcag aggccactgg agaatcagcg
aggacaccca gctgatggag 240ctggtgagcg tgtacggccc ccagaactgg
aaccacatcg ccgagagcat gcagggcaga 300accggcaaga gctgcagact
gagatggttc aaccagctgg accccagaat caacaagaga 360gccttcagcg
acgaggagga ggagagactg ctggccgccc acagagcctt cggcaacaag
420tgggccatga tcgccaagct gttcaacggc agaaccgaca acgccctgaa
gaaccactgg 480cacgtgctga tggccagaaa gatgagacag cagagcagca
gctacgtgca gagattcaac 540ggcagcgccc acgagagcaa cagcgaccac
aaggtgttca acctgagccc cggcaacgtg 600gacgaggacg aggacgtgaa
cggcaagaag tgcagctacg agatgctgaa ggagggcagc 660acccagctgc
acgcccagta cctgcaggag gactacacca gcagcagaat gcccgcccag
720ggcccccacc accactacac cacctggccc gccgacagcc tggccctgac
cctgcacgtg 780tgcatccagg agcccagcag cagcagcagc atcagcatcc
ccagcaccag caccaccggc 840gagcacacca tgctgaccag atacttcgag
accgtgaagc cccccgcctt catcgacttc 900ctgggcgtgg gccac
91550305PRTArtificial SequenceAmino acid sequence MybTF, variant 29
50Met Lys Met Asp Tyr Ser Cys Phe Gln Glu Tyr Pro Phe Asp Phe His 1
5 10 15 Cys Arg Ala Thr Thr Phe Asn Gly Phe His Glu Asn Asn Ala Val
Ser 20 25 30 Glu Thr Val Glu Glu Phe Cys Asn His Arg Arg Met Gln
Lys Lys Ser 35 40 45 Asp Asp Gly His Thr Lys Arg Lys Lys Arg Gln
Ser Val Ser Arg Val 50 55 60 Cys Ser Arg Gly His Trp Arg Ile Ser
Glu Asp Thr Gln Leu Met Glu 65 70 75 80 Leu Val Ser Val Tyr Gly Pro
Gln Asn Trp Asn His Ile Ala Glu Ser 85 90 95 Met Gln Gly Arg Thr
Gly Lys Ser Cys Arg Leu Arg Trp Phe Asn Gln 100 105 110 Leu Asp Pro
Arg Ile Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu 115 120 125 Arg
Leu Leu Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile 130 135
140 Ala Lys Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp
145 150 155 160 His Val Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser
Ser Tyr Val 165 170 175 Gln Arg Phe Asn Gly Ser Ala His Glu Ser Asn
Ser Asp His Lys Val 180 185 190 Phe Asn Leu Ser Pro Gly Asn Val Asp
Glu Asp Glu Asp Val Asn Gly 195 200 205 Lys Lys Cys Ser Tyr Glu Met
Leu Lys Glu Gly Ser Thr Gln Leu His 210 215 220 Ala Gln Tyr Leu Gln
Glu Asp Tyr Thr Ser Ser Arg Met Pro Ala Gln 225 230 235 240 Gly Pro
His His His Tyr Thr Thr Trp Pro Ala Asp Ser Leu Ala Leu 245 250 255
Thr Leu His Val Cys Ile Gln Glu Pro Ser Ser Ser Ser Ser Ile Ser 260
265 270 Ile Pro Ser Thr Ser Thr Thr Gly Glu His Thr Met Leu Thr Arg
Tyr 275 280 285 Phe Glu Thr Val Lys Pro Pro Ala Phe Ile Asp Phe Leu
Gly Val Gly 290 295 300 His 305 51915DNAArtificial
SequenceSynthetic polynucleotide 51atgaagatgg acttcagctg cttccaggag
taccccttcg agttccactg cagaggctgc 60accttcaacg gcttcagaga gaacaacgcc
gtgagcgaca ccgtggagga gttctgccag 120aagagaaaga tgcagaagaa
gtgcgacgac ctgagaacca agaagaagaa gaagcagagc 180gtgagcagag
tgtgcagcag aggccactgg agaatcagcg aggacaccca gctgatggag
240ctggtgagcg tgtacggccc ccagaactgg aaccacatcg ccgagagcat
gcagggcaga 300accggcaaga gctgcagact gagatggttc aaccagctgg
accccagaat caacaagaga 360gccttcagcg acgaggagga ggagagactg
ctggccgccc acagagcctt cggcaacaag 420tgggccatga tcgccaagct
gttcaacggc agaaccgaca acgccctgaa gaaccactgg 480cacgtgctga
tggccagaaa gatgagacag cagagcagca gctacgtgca gagattcaac
540ggcagcgccc acgagagcaa caccgaccac aagatcttcc agctgagccc
cggcaacgtg 600gacgacgacg aggacgtgca gctgaagaag tgcacctggg
agatgctgag agacggcacc 660accaacctga aggcccagta cctgaacgag
gagtacacca gcagcagaat gcccatgaac 720ggcccccacc accactacag
caccttcccc gccgagagcc tggccatcac cctgcacgtg 780agcgtgcagg
agcccagcac cagcagctgc ctgagcctgc ccagcagcag ctgcaccgcc
840gagcacaccc tggtgaccag atacttcgag accatcaagc cccccgcctt
catcgacttc 900ctgggcgtgg gcaga 91552305PRTArtificial SequenceAmino
acid sequence MybTF, variant 30 52Met Lys Met Asp Phe Ser Cys Phe
Gln Glu Tyr Pro Phe Glu Phe His 1 5 10 15 Cys Arg Gly Cys Thr Phe
Asn Gly Phe Arg Glu Asn Asn Ala Val Ser 20 25 30 Asp
Thr Val Glu Glu Phe Cys Gln Lys Arg Lys Met Gln Lys Lys Cys 35 40
45 Asp Asp Leu Arg Thr Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val
50 55 60 Cys Ser Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu
Met Glu 65 70 75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn Trp Asn His
Ile Ala Glu Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys Ser Cys Arg
Leu Arg Trp Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile Asn Lys Arg
Ala Phe Ser Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu Ala Ala His
Arg Ala Phe Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala Lys Leu Phe
Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp 145 150 155 160 His
Val Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val 165 170
175 Gln Arg Phe Asn Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile
180 185 190 Phe Gln Leu Ser Pro Gly Asn Val Asp Asp Asp Glu Asp Val
Gln Leu 195 200 205 Lys Lys Cys Thr Trp Glu Met Leu Arg Asp Gly Thr
Thr Asn Leu Lys 210 215 220 Ala Gln Tyr Leu Asn Glu Glu Tyr Thr Ser
Ser Arg Met Pro Met Asn 225 230 235 240 Gly Pro His His His Tyr Ser
Thr Phe Pro Ala Glu Ser Leu Ala Ile 245 250 255 Thr Leu His Val Ser
Val Gln Glu Pro Ser Thr Ser Ser Cys Leu Ser 260 265 270 Leu Pro Ser
Ser Ser Cys Thr Ala Glu His Thr Leu Val Thr Arg Tyr 275 280 285 Phe
Glu Thr Ile Lys Pro Pro Ala Phe Ile Asp Phe Leu Gly Val Gly 290 295
300 Arg 305 53915DNAArtificial SequenceSynthetic polynucleotide
53atgaagatgg agttcagctg cttccaggag ttccccttcg actggcactg caagggcacc
60accttccagg gcttcagaga gcagaacgcc gtgagcgaga ccgtggagga gttctgcaac
120aagagaagaa tgcagaagaa gagcgacgac ctgaagacca agagaaagaa
gaagcagagc 180gtgagcagag tgtgcagcag aggccactgg agaatcagcg
aggacaccca gctgatggag 240ctggtgagcg tgtacggccc ccagaactgg
aaccacatcg ccgagagcat gcagggcaga 300accggcaaga gctgcagact
gagatggttc aaccagctgg accccagaat caacaagaga 360gccttcagcg
acgaggagga ggagagactg ctggccgccc acagagcctt cggcaacaag
420tgggccatga tcgccaagct gttcaacggc agaaccgaca acgccctgaa
gaaccactgg 480cacgtgctga tggccagaaa gatgagacag cagagcagca
gctacgtgca gagattcaac 540ggcagcgccc acgagagcaa caccgacaga
agaatcttca acctgagccc cggccaggtg 600gacgacgacg aggacgtgaa
cctgaagaag tgcagctggg agatgctgaa ggagggcacc 660accaacctga
aggcccagtt cctgcaggag gagtacagca gcagcagaat gcccatgcag
720ggcccccacc accactacag caccttcccc gccgacagcc tggccctgag
cctgagagtg 780agcatccagg agcccagcag cagcagcagc ctgagcctgc
ccagcagctg caccaccggc 840gagcacacca tggtgaccag atacttcgag
agcatcaagc cccccgcctt catcgacttc 900ctgggcgtgg gccac
91554305PRTArtificial SequenceAmino acid sequence MybTF, variant 31
54Met Lys Met Glu Phe Ser Cys Phe Gln Glu Phe Pro Phe Asp Trp His 1
5 10 15 Cys Lys Gly Thr Thr Phe Gln Gly Phe Arg Glu Gln Asn Ala Val
Ser 20 25 30 Glu Thr Val Glu Glu Phe Cys Asn Lys Arg Arg Met Gln
Lys Lys Ser 35 40 45 Asp Asp Leu Lys Thr Lys Arg Lys Lys Lys Gln
Ser Val Ser Arg Val 50 55 60 Cys Ser Arg Gly His Trp Arg Ile Ser
Glu Asp Thr Gln Leu Met Glu 65 70 75 80 Leu Val Ser Val Tyr Gly Pro
Gln Asn Trp Asn His Ile Ala Glu Ser 85 90 95 Met Gln Gly Arg Thr
Gly Lys Ser Cys Arg Leu Arg Trp Phe Asn Gln 100 105 110 Leu Asp Pro
Arg Ile Asn Lys Arg Ala Phe Ser Asp Glu Glu Glu Glu 115 120 125 Arg
Leu Leu Ala Ala His Arg Ala Phe Gly Asn Lys Trp Ala Met Ile 130 135
140 Ala Lys Leu Phe Asn Gly Arg Thr Asp Asn Ala Leu Lys Asn His Trp
145 150 155 160 His Val Leu Met Ala Arg Lys Met Arg Gln Gln Ser Ser
Ser Tyr Val 165 170 175 Gln Arg Phe Asn Gly Ser Ala His Glu Ser Asn
Thr Asp Arg Arg Ile 180 185 190 Phe Asn Leu Ser Pro Gly Gln Val Asp
Asp Asp Glu Asp Val Asn Leu 195 200 205 Lys Lys Cys Ser Trp Glu Met
Leu Lys Glu Gly Thr Thr Asn Leu Lys 210 215 220 Ala Gln Phe Leu Gln
Glu Glu Tyr Ser Ser Ser Arg Met Pro Met Gln 225 230 235 240 Gly Pro
His His His Tyr Ser Thr Phe Pro Ala Asp Ser Leu Ala Leu 245 250 255
Ser Leu Arg Val Ser Ile Gln Glu Pro Ser Ser Ser Ser Ser Leu Ser 260
265 270 Leu Pro Ser Ser Cys Thr Thr Gly Glu His Thr Met Val Thr Arg
Tyr 275 280 285 Phe Glu Ser Ile Lys Pro Pro Ala Phe Ile Asp Phe Leu
Gly Val Gly 290 295 300 His 305 55915DNAArtificial
SequenceSynthetic polynucleotide 55atgaagatgg acttcagctg cttccaggag
taccccttcg agttccactg cagaggcacc 60accttcaacg gctggagaga gaacaacgcc
gtgagcgaga ccgtggagga gttcacccag 120agaagaagaa tgcagaagaa
gaccgacgac ctgaagacca agaagaagaa gaagcagagc 180gtgagcagag
tgtgcagcag aggccactgg agaatcagcg aggacaccca gctgatggag
240ctggtgagcg tgtacggccc ccagaactgg aaccacatcg ccgagagcat
gcagggcaga 300accggcaaga gctgcagact gagatggttc aaccagctgg
accccagaat caacaagaga 360gccttcagcg acgaggagga ggagagactg
ctggccgccc acagagcctt cggcaacaag 420tgggccatga tcgccaagct
gttcaacggc agaaccgaca acgccctgaa gaaccactgg 480cacgtgctga
tggccagaaa gatgagacag cagagcagca gctacgtgca gagattcaac
540ggcagcgccc acgagagcaa caccgaccac aagatcttca acctgacccc
cggcaacgtg 600gacgacgacg aggacgtgaa cctgaagaag tgcagctggg
agatgctgaa ggagggcacc 660accaacctga aggcccagta cctgcaggac
gagtacagca gcagcagaat gcccatgcag 720ggcccccacc accactacag
caccttcccc gccgacagcc tggccctgac cctgcacgtg 780agcatccagg
agcccagcag caccagcagc ctgagcctgc ccaccagcag caccaccggc
840gagcacacca tggtgaccag atacttcgag accatcaagc cccccgcctt
catcgacttc 900ctgggcgtgg gccac 91556305PRTArtificial SequenceAmino
acid sequence MybTF, variant 32 56Met Lys Met Asp Phe Ser Cys Phe
Gln Glu Tyr Pro Phe Glu Phe His 1 5 10 15 Cys Arg Gly Thr Thr Phe
Asn Gly Trp Arg Glu Asn Asn Ala Val Ser 20 25 30 Glu Thr Val Glu
Glu Phe Thr Gln Arg Arg Arg Met Gln Lys Lys Thr 35 40 45 Asp Asp
Leu Lys Thr Lys Lys Lys Lys Lys Gln Ser Val Ser Arg Val 50 55 60
Cys Ser Arg Gly His Trp Arg Ile Ser Glu Asp Thr Gln Leu Met Glu 65
70 75 80 Leu Val Ser Val Tyr Gly Pro Gln Asn Trp Asn His Ile Ala
Glu Ser 85 90 95 Met Gln Gly Arg Thr Gly Lys Ser Cys Arg Leu Arg
Trp Phe Asn Gln 100 105 110 Leu Asp Pro Arg Ile Asn Lys Arg Ala Phe
Ser Asp Glu Glu Glu Glu 115 120 125 Arg Leu Leu Ala Ala His Arg Ala
Phe Gly Asn Lys Trp Ala Met Ile 130 135 140 Ala Lys Leu Phe Asn Gly
Arg Thr Asp Asn Ala Leu Lys Asn His Trp 145 150 155 160 His Val Leu
Met Ala Arg Lys Met Arg Gln Gln Ser Ser Ser Tyr Val 165 170 175 Gln
Arg Phe Asn Gly Ser Ala His Glu Ser Asn Thr Asp His Lys Ile 180 185
190 Phe Asn Leu Thr Pro Gly Asn Val Asp Asp Asp Glu Asp Val Asn Leu
195 200 205 Lys Lys Cys Ser Trp Glu Met Leu Lys Glu Gly Thr Thr Asn
Leu Lys 210 215 220 Ala Gln Tyr Leu Gln Asp Glu Tyr Ser Ser Ser Arg
Met Pro Met Gln 225 230 235 240 Gly Pro His His His Tyr Ser Thr Phe
Pro Ala Asp Ser Leu Ala Leu 245 250 255 Thr Leu His Val Ser Ile Gln
Glu Pro Ser Ser Thr Ser Ser Leu Ser 260 265 270 Leu Pro Thr Ser Ser
Thr Thr Gly Glu His Thr Met Val Thr Arg Tyr 275 280 285 Phe Glu Thr
Ile Lys Pro Pro Ala Phe Ile Asp Phe Leu Gly Val Gly 290 295 300 His
305
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References